High throughput method and system for screening candidate compounds for activity against target ion channels

ABSTRACT

Drug candidate screening methods are applied to discover compounds with activity against ion channel targets. The method may include modulating the transmembrane potential of host cells in a plurality of sample wells with a repetitive application of electric fields so as to set the transmembrane potential to a level corresponding to a pre-selected voltage dependent state of a target ion channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/804,580, filed on Mar. 12, 2001 now U.S. Pat. No. 6,686,193, entitledHIGH THROUGHPUT METHOD AND SYSTEM FOR SCREENING CANDIDATE COMPOUNDS FORACTIVITY AGAINST TARGET ION CHANNELS which application further claimspriority under 35 U.S.C. Section 119(e) to U.S. Provisional ApplicationSerial No. 60/217,671, entitled INSTRUMENTATION AND METHODS FORELECTRICAL STIMULATION, filed on Jul. 10, 2000, both of which are herebyincorporated by reference in their entireties. This application is alsorelated to the following three additional U.S. Patent Applications, alsoincorporated by reference to this application in their entireties:application Ser. No. 09/804,457, entitled ION CHANNEL ASSAY METHODS,filed Mar. 12, 2001; application Ser. No. 09/804,480, entitled IONCHANNEL ASSAY METHODS, filed Mar. 12, 2001; and application Ser. No.09/804,458, entitled MULTI-WELL PLATE AND ELECTRODE ASSEMBLIES FOR IONCHANNEL ASSAYS, filed Mar. 12, 2001

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to instrumentation and methodsfor manipulating membrane potentials of living cells via electricalstimulation.

2. Description of the Related Art

It has long been known that the interior of animal and plant cells iselectrically negative with respect to the exterior. The magnitude ofthis potential difference is generally between 5 and 90 mV, with most ofthe potential being developed across the cell membrane. Thetransmembrane potential of a given cell is set by the balance of theactivities of ion transporters which create and maintain theelectrochemical gradient, and the activities of ion channels, passivediffusion and other factors, that allow ions to flow through the plasmamembrane.

Ion channels participate in, and regulate, cellular processes as diverseas the generation and timing of action potentials, energy production,synaptic transmission, secretion of hormones and the contraction ofmuscles, etc. Many drugs exert their specific effects via modulation ofion channels. Examples include antiepileptic compounds like phenyloinand lamotrigine, which block voltage-dependent sodium channels in thebrain, antihypertensive drugs like nifedipine and diltiazem, which blockvoltage-dependent calcium channels in smooth muscle cells, andstimulators of insulin release like glibenclamide and tolbutamide, whichblock ATP-regulated potassium channels in the pancreas.

Finding new drugs which have specific modulatory effects on ion channelsrequires methods for measuring and manipulating the membrane potentialof cells with the ion channels present in the membrane. A number ofmethods exist today that can be used to measure cell transmembranepotentials and to measure the activities of specific ion channels.Probably the best known approach is the patch clamp, originallydeveloped by Neher, Sakmann, and Steinback. (The Extracellular PatchClamp, A Method For Resolving Currents Through Individual Open ChannelsIn Biological Membranes”, Pfluegers Arch. 375; 219-278, 1978). Othermethods include optical recording of voltage-sensitive dyes (Cohen etal., Annual Reviews of Neuroscience 1: 171-82, 1978) and extracellularrecording of fast events using metal (Thomas et al., Exp. Cell Res. 74:61-66, 1972) or field effect transistors (FET) (Fromherz et al., Science252: 1290-1293, 1991) electrodes.

The patch clamp technique allows measurement of ion flow through ionchannel proteins and the analysis of the effect of drugs on ion channelsfunction. In brief, in the standard patch clamp technique, a thin glasspipette is heated and pulled until it breaks, forming a very thin (<1 μmin diameter) opening at the tip. The pipette is filled with saltsolution approximating the intracellular ionic composition of the cell.A metal electrode is inserted into the large end of the pipette, andconnected to associated electronics. The tip of the patch pipette ispressed against the surface of the cell membrane. The pipette tip sealstightly to the cell and isolates a few ion channel proteins in a tinypatch of membrane. The activity of these channels can be measuredelectrically (single channel recording) or, alternatively, the patch canbe ruptured allowing measurements of the combined channel activity ofthe entire cell membrane (whole cell recording).

During both single channel recording and whole-cell recording, theactivity of individual channel subtypes can be further resolved byimposing a “voltage clamp” across the membrane. Through the use of afeedback loop, the “voltage clamp” imposes a user-specified potentialdifference across the membrane, allowing measurement of the voltage,ion, and time dependencies of various ion channel currents. Thesemethods allow resolution of discrete ion channel subtypes.

A major limitation of the patch clamp technique as a general method inpharmacological screening is its low throughput. Typically, a single,highly trained operator can test fewer than ten compounds per day usingthe patch clamp technique. Furthermore the technique is not easilyamenable to automation, and produces complex results that requireextensive analysis by skilled electrophysiologists. By comparison, theuse of optical detection systems provides for significantly greaterthroughput for screening applications (currently, up to 100,000compounds per day), while at the same time providing for highlysensitive analysis of transmembrane potential. Methods for the opticalsensing of membrane potential are typically based on translocation,redistribution, orientation changes, or shifts in spectra offluorescent, luminescent, or absorption dyes in response to the cellularmembrane potential (see generally González, et al., Drug Discovery Today4:431-439, 1999).

A preferred optical method of analysis has been previously described(González and Tsien, Chemistry and Biology 4: 269-277, 1997; Gonzálezand Tsien, Biophysical Journal 69: 1272-1280, 1995; and U.S. Pat. No.5,661,035 issued Aug. 26, 1997, hereby incorporated by reference). Thisapproach typically comprises two reagents that undergo energy transferto provide a ratiometric fluorescent readout that is dependent upon themembrane potential. The ratiometric readout provides importantadvantages for drug screening including improved sensitivity,reliability and reduction of many types of experimental artifacts.

Compared to the use of a patch clamp, optical methods of analysis do notinherently provide the ability to regulate, or clamp, the transmembranepotential of a cell. Clamping methods are highly desirable because theyprovide for significantly enhanced, and more flexible methods of ionchannel measurement. A need thus exists for reliable and specificmethods of regulating the membrane potentials of living cells that arecompatible with optical methods of analysis and are readily amendable tohigh throughput analysis.

SUMMARY OF THE INVENTION

Methods and systems of compound screening are provided. In oneembodiment, such a method comprises expressing the target ion channel ina population of host cells and placing a plurality of the host cellsinto each of a plurality of sample wells. A candidate drug compound isadded to at least one of the plurality of sample wells; and thetransmembrane potential of the cells is modulated with a repetitiveapplication of electric fields so as to set the transmembrane potentialto a level corresponding to a pre-selected voltage dependent state ofthe target ion channel. Apparatus for high throughput screening is alsoprovided. In one specific embodiment, a plurality of wells having a hightransmittance portion through which cells present in the wells areoptically observable in an area of observation are each provided withtwo electrodes. A power supply is connected to the electrodes; whereinthe power supply and the electrodes are configured to apply a series ofelectric fields to cells within the area of observation, the electricfields having a spatial variation of less than about 25% of a mean fieldintensity within the area of observation, the electric fields beingeffective to controllably alter the transmembrane potential of a portionof the cells. In addition, an optical detector is configured to detectlight emanating from the wells through the high transmittance portion,and a data processing unit is provided to interpret the light emanatingfrom the wells through the high transmittance portion as ion channelactivity resulting from the transmembrane potential alterations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows one embodiment of a dipper electrode array.

FIG. 2 Shows a number of embodiments of multiwell plates comprisingsurface electrodes.

FIG. 3 Shows a block diagram of one embodiment of the electricalstimulation system.

FIG. 4. Shows the simulated effects of repetitive external electricalfields on a cell expressing a voltage dependent sodium channel. Theupper panel indicates the applied electrical field, the middle panelindicates the simulated sodium current into the cell, and the lowerpanel indicates the simulated average transmembrane potential.

FIG. 5 Shows a schematic representation of a square wave.

FIG. 6 Shows examples of various wave kernels.

FIG. 7 Shows calculated electric field profiles for various electrodeassemblies in round, 6.2 mm diameter wells. Dashed circle is a 3 mmdiameter view window. In white areas, the electric field strength isless than 10% of the average electric field strength in the view window.In gray areas, the electric field strength is within 10% of the averageelectric field strength in the view window. In black areas, the electricfield strength is greater than 10% of the average electric fieldstrength in the view window.

FIG. 8 Shows calculated electric field profiles for various electrodeassemblies in round and square wells 6.2 mm across. Dashed circle is a 3mm diameter view window. In white areas, the electric field strength isless than 1% of the average electric field strength in the view window.In gray areas, the electric field strength is within 1% of the averageelectric field strength in the view window. In black areas, the electricfield strength is greater than 1% of the average electric field strengthin the view window.

FIG. 9 Shows various electrode and insulator designs for improvingelectric field uniformity in round wells.

FIG. 10 Shows the effect of electrical stimulation protocols at varyingpulse amplitudes over the time course of electrical stimulation inwild-type CHO cells.

FIG. 11 Shows the relationship between the maximal cellular response andthe applied pulse amplitude during electrical stimulation for wild-typeCHO cells. Data was from FIG. 10 taken after about 5 seconds.

FIG. 12 Shows the dose response curve for the effect of TTX in wild-typeCHO cells. Stimulation parameters were 33 V/cm, 50 Hz for 3 seconds witha biphasic square wave kernel (5 ms per phase). The solid line is a Hillfunction fit to the data with EC₅₀=9 nM and a Hill coefficient of 1.47.

FIG. 13 Shows the relationship between pulse duration and frequency andthe cellular response wild-type CHO cells during electrical stimulation.The electric field strength was always 25 V/cm. The stimulus was athree-second burst of biphasic pulses of varying duration and frequency.Solid lines are fits to the form

$R = {1 + {\frac{Af}{f + f_{0}}.}}$

FIG. 14 Shows time traces for CHO cells expressing the NaV2 sodiumchannel cells electrically stimulated at various field strengths. Cellswere stimulated in a 96-well plate, with a 20 Hz, 3 second-long train ofbiphasic, 5 ms/phase voltage pulses. The stimulation occurred during theshaded portion of the graph. In this experiment, the cells were stainedwith 20 μM CC2-DMPE and 63 nM DiSBAC₆(3). This dye combination has a 2ms time constant and accurately tracks the transmembrane potential. Therise and fall times of the response were fitted to exponential decayfunctions and were found to be τ_(rise)=200 ms and τ_(fall)=850 ms.

FIG. 15 Shows the relationship between the electric field strength andthe cellular response measured after 4 seconds (squares) and 10 seconds(circles) of electrical stimulation. The line is a Boltzman fit to thedata.

FIG. 16 Shows the effect of pulse duration and stimulation frequency onthe cellular response of CHO cells expressing the NaV2 sodium channel.

FIG. 17 Shows the knee time parameter T₀ from the fits to the data inFIG. 16 plotted versus the stimulus duration.

FIG. 18 Shows the temporal response of HEK-293 cells expressing the NaV3sodium channel during electrical stimulation.

FIG. 19 Shows dose response curves for tetracaine (FIG. 19A) andtetrodotoxin (FIG. 19B) for HEK-293 expressing the NaV3 sodium channel.Electrical stimulation conditions were: E=33 V/cm, 10 ms/phase biphasicstimulation, 15 Hz burst for 1.5 seconds.

FIG. 20 Shows a dose response curve for tetracaine for HEK-293expressing the NaV4 ion channel. For this experiment, electricalstimulation parameters were E=33 V/cm, 10 ms/phase biphasic stimulation,15 Hz burst for 1.5 seconds.

FIG. 21. Shows a full-plate view of electrical stimulation of wild-typeHEK-293 cells. Each individual panel represents the time trace of thenormalized fluorescence ratio of a single well in the 96-well plate.Each well in a vertical column was stimulated simultaneously with thesame field strength. Field strength increases from left to right. Rows6-8 contained 10 mM TEA to block the voltage-dependent potassiumchannels.

FIG. 22. Shows the cellular response as a function of the stimulus fieldfor wild type HEK. Error bars are standard deviations. Open symbols: noadded blockers. Filled symbols: 10 mM TEA added to block potassiumchannels.

FIG. 23 Shows the time response traces for selected concentrations ofthe sodium channel blockers tetrodoxin (TTX) (FIG. 23A) and tetracaine(FIG. 23B) in CHO cells expressing the NaV2 sodium channel.

FIG. 24 Shows the dose response curves for TTX and tetracaine inhibitionof the NaV2 sodium channel.

FIG. 25. Shows a ‘Random’ TTX spiking experiment. Each small box in this11×8 array contains the ten-second time trace of a well at thecorresponding position of a 96-well plate. The twelfth column was acontrol well without cells used for background subtraction and is notshown. Wells (1,1), (2,2), (3,3), etc. contained a blockingconcentration of TTX.

FIG. 26 Shows an analysis of the ‘random’ TTX spiking data shown in FIG.25. The data points are the ratiometric response in the time window from1.8-2.4 seconds after the beginning of the stimulus burst (i.e. at thepeak of the response). The filled circles points were spiked with 1 μMTTX; the open circles had no blocker added.

FIG. 27. Shows a full-plate view of electrically-stimulated HL5 cardiacmuscle cells. Each individual panel represents the time trace of thenormalized fluorescence ratio of a single well in the 96-well plate.Each well in a vertical column was stimulated simultaneously with thesame field strength. Field strength increases from left to right. Rows 5and 6 contained 10 μM TTX to partially block the voltage-dependentsodium channels. Rows 7 and 8 contained 10 mM TEA to partially block thevoltage-dependent potassium channels.

FIG. 28. Shows the response of HL5 cells as a function of the appliedelectric field strength. Black points are the average of the response offour wells with no added compounds. The solid line is a Boltzman fit tothe data with E₅₀=22 V/cm. The points are the screening window: thedifference between the response and the unstimulated response normalizedto the standard deviation of the response (see Appendix A3).

FIG. 29 The typical voltage response for CHO cells expressing apotassium channel and the NaV3 sodium channel after a three separatestimulation cycles using surface electrodes.

FIG. 30 Shows the average ratiometric response of a population of cellsgrown in a 96 well multiwell plate stimulated with monophasic stimuli ofvarying field strengths via surface electrodes. The points in this curveare the average peak response of 4 stimulations on the same culture.

FIG. 31. Shows the cellular response as a function of the stimulus fieldfor wild type RBL. Error bars are standard deviations. Open symbols: noadded blockers. Filled symbols: 400 μM TEA added to block IRK1 channels.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Generally, the nomenclature used herein and many of the fluorescence,computer, detection, chemistry and laboratory procedures described beloware those well known and commonly employed in the art. Standardtechniques are usually used for chemical synthesis, fluorescence,optics, molecular biology, computer software and integration. Generally,chemical reactions, cell assays and enzymatic reactions are performedaccording to the manufacturer's specifications where appropriate. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references,including those listed below, which are herein incorporated byreference.

-   -   Lakowicz, J. R. Topics in Fluorescence Spectroscopy, (3 volumes)        New York: Plenum Press (1991), and Lakowicz, J. R. Emerging        applications of fluorescence spectroscopy to cellular imaging:        lifetime imaging, metal-ligand probes, multi-photon excitation        and light quenching. Scanning Microsc Suppl Vol. 10 (1996) pages        213-24, for fluorescence techniques;    -   Sambrook et al. Molecular Cloning: A Laboratory Manual, 2^(nd)        ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring        Harbor, N.Y., for molecular biology methods;    -   Cells: A Laboratory Manual, 1^(st) edition (1998) Cold Spring        Harbor Laboratory Press, Cold Spring Harbor, N.Y., for cell        biology methods;    -   Optics Guide 5 Melles Griot® Irvine Calif., Optical Waveguide        Theory, Snyder & Love published by Chapman & Hall for general        optical methods;    -   Hille, B. Ionic Channels of Excitable membranes, Second        Edition (1992) Sinauer Associates, Inc., Sunderland, Mass. for        general electrophysiological methods and properties of ion        channels.    -   Horowitz and Hill, The Art of Electronics, Second Edition (1989)        Cambridge University Press, Cambridge, U.K. for electronic        circuits.        The following definitions are set forth to illustrate and define        the meaning and scope of the various terms used to describe the        invention herein.

-   The term “activation” refers to the transition from a resting    (non-conducting) state of an ion channel to the activated    (conducting) state.

-   The term “activation threshold” refers to the lowest potential above    which measurable opening of a channel occurs.

-   The term “anode” refers to an electrode when driven to a positive    potential relative to earth by an external source.

-   The term “area of cellular stimulation” means the area defined by    two electrodes that experiences significant electrical stimulation    (typically 5V/cm or higher) in which the cells of interest are    located. Typically the area of cellular stimulation is larger than,    or equal to, the area of observation. For standard 96-well based    measurements the area of cellular stimulation is typically about 16    mm².

-   The term “area of observation” means the portion of the system over    which a measurement is taken. The area of observation is typically    an area of at least 0.5 mm² for multiwell plate based measurements.

-   The term “bioluminescent protein” refers to a protein capable of    causing the emission of light through the catalysis of a chemical    reaction. The term includes proteins that catalyze bioluminescent or    chemiluminescent reactions, such as those causing the oxidation of    luciferins. The term “bioluminescent protein” includes not only    bioluminescent proteins that occur naturally, but also mutants that    exhibit altered spectral or physical properties.

-   The term “biphasic” refers to a pulse with two parts, each with an    opposite polarity.

-   The term “Boltzman function” refers to the sigmoidal (i.e.    step-like) response function

${y(x)} = {y_{0} + {\frac{A}{1 + {\exp\left( \frac{x - x_{50}}{\Delta\; x} \right)}}.}}$Where: y is the independent variable

-   -   y₀ is an adjustable parameter equal to the limit of the function        as x→∞    -   A is an adjustable parameter equal to step size    -   x₅₀ is an adjustable parameter related to the midpoint of the        step    -   Δx is an adjustable parameter describing the width of the step

-   The term “cathode” refers to an electrode when driven to a negative    potential relative to earth by an external source.

-   The term “depolarize” means to cause the transmembrane potential of    a cell to become closer to zero. In the case of cells that are    normally at negative resting potentials, this term means that the    transmembrane potential changes in a positive direction.

-   The term “effective concentration (50%)” or “EC₅₀” refers to the    concentration at which a pharmacological compound has half the    effectiveness compared to the maximal effectiveness at high    concentrations of the compound.

-   The term “electrically excitable” refers to a cell or tissue that    responds to a suprathreshold electrical stimulus by generating an    action potential. Electrically excitable cells contain at least one    voltage-dependent ion channel type generating an inward current and    at least one ion channel type generating an outward current.

-   The term “electrical stimulation” means initiating a voltage change    in cells using an extracellular current pulse.

-   The term “electrode” means a controllable conductive interface    between an instrument and a test system.

-   The term “electropermeablization” refers to mild electroporation, in    which the hydrated pores created through the membrane are only large    enough to pass water molecules and small single-atom ions.

-   The term “electroporation” refers to a phenomenon in which the    application of a large electric potential across the membrane of a    cell results in dielectric breakdown of the membrane, and the    creation of hydrated pathways through the membrane.

-   The term “fluorescent component” refers to a component capable of    absorbing light and then re-emitting at least some fraction of that    energy as light over time. The term includes discrete compounds,    molecules, naturally fluorescent proteins and marco-molecular    complexes or mixtures of fluorescent and non-fluorescent compounds    or molecules. The term “fluorescent component” also includes    components that exhibit long lived fluorescence decay such as    lanthanide ions and lanthanide complexes with organic ligand    sensitizers, that absorb light and then re-emit the energy over    milliseconds.

-   The term “FRET” refers to fluorescence resonance energy transfer.    For the purposes of this invention, FRET includes energy transfer    processes that occur between two fluorescent components, a    fluorescent component and a non-fluorescent component, a luminescent    component and a fluorescent component and a luminescent component    with a non-fluorescent component.

-   The term “gene knockout” as used herein, refers to the targeted    disruption of a gene in vivo with complete loss of function that has    been achieved by any transgenic technology familiar to those in the    art. In one embodiment, transgenic animals having gene knockouts are    those in which the target gene has been rendered nonfunctional by an    insertion targeted to the gene to be rendered non-functional by    homologous recombination.

-   The term “Hill function” refers to the sigmoidal (i.e. step-like)    response function

${y(x)} = {y_{0} + {\frac{A}{x_{0}^{n} + x^{n}}.}}$Where: y is the independent variable

-   -   y₀ is an adjustable parameter equal to the limit of the function        as x→∞    -   A is an adjustable parameter equal to step size    -   x₀ is an adjustable parameter related to the midpoint of the        step    -   n is an adjustable parameter describing the steepness of the        step

-   The term “Hill coefficient” refers to the parameter n in the Hill    function.

-   The term “hit” refers to a test compound that shows desired    properties in an assay.

-   The term “homolog” refers to two sequences or parts thereof, that    are greater than, or equal to 75% identical when optimally aligned    using the ALIGN program. Homology or sequence identity refers to the    following. Two amino acid sequences are homologous if there is a    partial or complete identity between their sequences. For example,    85% homology means that 85% of the amino acids are identical when    the two sequences are aligned for maximum matching. Gaps (in either    of the two sequences being matched) are allowed in maximizing    matching; gap lengths of 5 or less are preferred with 2 or less    being more preferred. Alternatively and preferably, two protein    sequences (or polypeptide sequences derived from them of at least 30    amino acids in length) are homologous, as this term is used herein,    if they have an alignment score of more than 5 (in standard    deviation units) using the program ALIGN with the mutation data    matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in    Atlas of Protein Sequence and Structure, 1972, volume 5, National    Biomedical Research Foundation, pp. 101-110, and Supplement 2 to    this volume, pp. 1-10.

-   The term “hyperpolarize” means to cause the transmembrane potential    of a cell to move farther away from zero. In the case of cells that    are normally at negative resting potentials, this term means that    the transmembrane potential changes in a negative direction.

-   The term “inactivation” means that an ion channel moves into the    inactivated state.

-   The term “inactivated” refers to a voltage-dependent ion channel in    a particular non-conducting conformational state. Transitions into    and out of the inactivated state are generally slow relative to    transitions between other conformational states. The inactivated    state is usually the preferred state at elevated transmembrane    potentials. At low transmembrane potentials, the inactivated state    is unstable and relaxes to the resting state.

-   The term “kernel” means a mathematical function intended to be    convoluted with one or more other time-varying functions. In theory,    the kernel can be any function that tends to zero as the independent    variable tends to ±∞. In practice, the kernel can be any waveform    that can programmed into an arbitrary wavefunction generator, or    that can be generated by a computer-controlled digital to analog    (D/A) converter.

-   The term “luminescent component” refers to a component capable of    absorbing energy, such as electrical (e.g. Electro-luminescence),    chemical (e.g. chemi-luminescence) or acoustic energy and then    emitting at least some fraction of that energy as light over time.

-   The term “component” includes discrete compounds, molecules,    bioluminescent proteins and macro-molecular complexes or mixtures of    luminescent and non-luminescent compounds or molecules that act to    cause the emission of light.

-   The term “transmembrane potential modulator” refers to components    capable of altering the resting or stimulated transmembrane    potential of a cellular or sub-cellular compartment. The term    includes discrete compounds, ion channels, receptors, pore forming    proteins, or any combination of these components.

-   The term “membrane time constant” or “τ_(M)” means the product of    the membrane resistance (R_(M)) and capacitance (C_(M)).

-   The term “monophasic” refers to a pulse whose polarity does not    change to the opposite polarity.

-   The term “naturally fluorescent protein” refers to a protein capable    of forming a highly fluorescent, intrinsic chromophore either    through the cyclization and oxidation of internal amino acids within    the protein or via the enzymatic addition of a fluorescent    co-factor. The term includes wild-type fluorescent proteins and    engineered mutants that exhibit altered spectral or physical    properties. The term does not include proteins that exhibit weak    fluorescence by virtue only of the fluorescence contribution of    non-modified tyrosine, tryptophan, histidine and phenylalanine    groups within the protein.

-   The term “naturally occurring” refers to a component produced by    cells in the absence of artificial genetic or other modifications of    those cells.

-   The term “Multiwell plate” refers to a two dimensional array of    addressable wells located on a substantially flat surface. Multiwell    plates may comprise any number of discrete addressable wells, and    comprise addressable wells of any width or depth. Common examples of    multiwell plates include 96 well plates, 384 well plates and 3456    well Nanoplates™.

-   The term “operably linked” refers to a juxtaposition wherein the    components so described are in a relationship permitting them to    function in their intended manner. A control sequence “operably    linked” to a coding sequence is ligated in such a way that    expression of the coding sequence is achieved under conditions    compatible with the control sequences.

-   The term “polarized cell” means a cell with an electric potential    difference across its cell membrane.

-   The term “rectification” means that the conductance is non-linear,    with a preferred direction.

-   The term “release from inactivation” refers to the conversion of an    inactivated closed channel, to a resting closed channel that is now    capable of opening.

-   The term “repetitive” means to repeat at least twice.

-   The term “repolarize” means to cause the transmembrane potential of    a cell to approach its resting potential.

-   The term “resting” or “resting state” refers to a voltage-dependent    ion channel that is closed, but free from inactivation.

-   The term “resting potential” for a cell means the equilibrium    transmembrane potential of a cell when not subjected to external    influences.

-   The term “reversal potential” for a particular ion refers to the    transmembrane potential for which the inward and outward fluxes of    that ion are equal.

-   The term “substantially parallel” means that the distance between    the surfaces of two objects facing each other varies by less than    10%, preferably less than 5%, when measured at every point on the    relevant surface of each object.

-   The term “targetable” refers to a component that has the ability to    be localized to a specific location under certain conditions. For    example, a protein that can exist at two or more locations that has    the ability to translocate to a defined site under some condition(s)    is targetable to that site. Common examples include the    translocation of protein kinase C to the plasma membrane upon    cellular activation, and the binding of SH2 domain containing    proteins to phosphorylated tyrosine residues. The term includes    components that are persistently associated with one specific    location or site, under most conditions.

-   The term “threshold electroporation potential” refers to the    externally applied field strength above which detectable    electroporation of a living cell occurs.

-   The term “test compound” refers to a chemical to be tested by one or    more screening method(s) of the invention as a putative modulator. A    test compound can be any chemical, such as an inorganic chemical, an    organic chemical, a protein, a peptide, a carbohydrate, a lipid, or    a combination thereof. Usually, various predetermined concentrations    of test compounds are used for screening, such as 0.01 micromolar, 1    micromolar and 10 micromolar. Test compound controls can include the    measurement of a signal in the absence of the test compound or    comparison to a compound known to modulate the target.

-   The term “transformed” refers to a cell into which (or into an    ancestor of which) has been introduced, by means of recombinant    nucleic acid techniques, a heterologous nucleic acid molecule.

-   The term “transgenic” is used to describe an organism that includes    exogenous genetic material within all of its cells. The term    includes any organism whose genome has been altered by in vitro    manipulation of the early embryo or fertilized egg or by any    transgenic technology to induce a specific gene knockout.

-   The term “transgene” refers any piece of DNA which is inserted by    artifice into a cell, and becomes part of the genome of the organism    (i.e., either stably integrated or as a stable extrachromosomal    element) which develops from that cell. Such a transgene may include    a gene which is partly or entirely heterologous (i.e., foreign) to    the transgenic organism, or may represent a gene homologous to an    endogenous gene of the organism. Included within this definition is    a transgene created by the providing of an RNA sequence that is    transcribed into DNA and then incorporated into the genome. The    transgenes of the invention include DNA sequences that encode the    fluorescent or bioluminescent protein that may be expressed in a    transgenic non-human animal.

-   The term “transistor-transistor logic” or “TTL” refers to an    electronic logic system in which a voltage around +5V is TRUE and a    voltage around 0V is FALSE.

-   A “uniform electric field” means that the electric field varies by    no more than 15% from the mean intensity within the area of    observation at any one time.

-   The term “voltage sensor” includes FRET based voltage sensors,    electrochromic transmembrane potential dyes, transmembrane potential    redistribution dyes, extracellular electrodes, field effect    transistors, radioactive ions, ion sensitive fluorescent or    luminescent dyes, and ion sensitive fluorescent or luminescent    proteins, that are capable of providing an indication of the    transmembrane potential.

-   The following terms are used to describe the sequence relationships    between two or more polynucleotides: “reference sequence”,    “comparison window”, “sequence identity”, “percentage identical to a    sequence”, and “substantial identity”. A “reference sequence” is a    defined sequence used as a basis for a sequence comparison; a    reference sequence may be a subset of a larger sequence, for    example, as a segment of a full-length cDNA or gene sequence, or may    comprise a complete cDNA or gene sequence. Generally, a reference    sequence is at least 20 nucleotides in length, frequently at least    25 nucleotides in length, and often at least 50 nucleotides in    length. Since two polynucleotides may each (1) comprise a sequence    (i.e., a portion of the complete polynucleotide sequence) that is    similar between the two polynucleotides, and (2) may further    comprise a sequence that is divergent between the two    polynucleotides, sequence comparisons between two (or more)    polynucleotides are typically performed by comparing sequences of    the two polynucleotides over a “comparison window” to identify and    compare local regions of sequence similarity. A “comparison window”,    as used herein, refers to a conceptual segment of at least 20    contiguous nucleotide positions wherein a polynucleotide sequence    may be compared to a reference sequence of at least 20 contiguous    nucleotides and wherein the portion of the polynucleotide sequence    in the comparison window may comprise additions or deletions (i.e.,    gaps) of 20 percent or less as compared to the reference sequence    (which does not comprise additions or deletions) for optimal    alignment of the two sequences. Optimal alignment of sequences for    aligning a comparison window may be conducted by the local homology    algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by    the homology alignment algorithm of Needleman and Wunsch (1970) J.    Mol. Biol. 48: 443, by the search for similarity method of Pearson    and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by    computerized implementations of these algorithms (GAP, BESTFIT,    FASTA, and TFASTA in the Wisconsin Genetics Software Package Release    7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by    inspection, and the best alignment (i.e., resulting in the highest    percentage of homology over the comparison window) generated by the    various methods is selected. The term “sequence identity” means that    two polynucleotide sequences are identical (i.e., on a    nucleotide-by-nucleotide basis) over the window of comparison. The    term “percentage identical to a sequence” is calculated by comparing    two optimally aligned sequences over the window of comparison,    determining the number of positions at which the identical nucleic    acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to    yield the number of matched positions, dividing the number of    matched positions by the total number of positions in the window of    comparison (i.e., the window size), and multiplying the result by    100 to yield the percentage of sequence identity. The terms    “substantial identity” as used herein denotes a characteristic of a    polynucleotide sequence, wherein the polynucleotide comprises a    sequence that has at least 30 percent sequence identity, preferably    at least 50 to 60 percent sequence identity, more usually at least    60 percent sequence identity as compared to a reference sequence    over a comparison window of at least 20 nucleotide positions,    frequently over a window of at least 25-50 nucleotides, wherein the    percentage of sequence identity is calculated by comparing the    reference sequence to the polynucleotide sequence which may include    deletions or additions which total 20 percent or less of the    reference sequence over the window of comparison.

-   As applied to polypeptides, the term “substantial identity” means    that two peptide sequences, when optimally aligned, such as by the    programs GAP or BESTFIT using default gap weights, share at least 30    percent sequence identity, preferably at least 40 percent sequence    identity, more preferably at least 50 percent sequence identity, and    most preferably at least 60 percent sequence identity. Preferably,    residue positions which are not identical differ by conservative    amino acid substitutions. Conservative amino acid substitutions    refer to the interchangeability of residues having similar side    chains. For example, a group of amino acids having aliphatic side    chains is glycine, alanine, valine, leucine, and isoleucine; a group    of amino acids having aliphatic-hydroxyl side chains is serine and    threonine; a group of amino acids having amide-containing side    chains is asparagine and glutamine; a group of amino acids having    aromatic side chains is phenylalanine, tyrosine, and tryptophan; a    group of amino acids having basic side chains is lysine, arginine,    and histidine; and a group of amino acids having sulfur-containing    side chains is cysteine and methionine. Preferred conservative amino    acids substitution groups are: valine-leucine-isoleucine,    phenylalanine-tyrosine, lysine-arginine, alanine-valine,    glutamic-aspartic, and asparagine-glutamine.

Since the list of technical and scientific terms cannot be allencompassing, any undefined terms shall be construed to have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs. Furthermore, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. For example, reference to a “restriction enzyme” or a “highfidelity enzyme” may include mixtures of such enzymes and any otherenzymes fitting the stated criteria, or reference to the method includesreference to one or more methods for obtaining cDNA sequences which willbe known to those skilled in the art or will become known to them uponreading this specification.

1. I Introduction

The present invention recognizes for the first time that thetransmembrane potentials of intact living cells comprising at least onevoltage regulated ion channel, can be precisely modulated via theapplication of repetitive electrical stimulation pulses to the fluidbathing the cells. The present invention includes instrumentation andmethods that provide for the accurate and reliable modulation of thetransmembrane potentials of intact living cells without significantlydisrupting their native cellular integrity.

As a non-limiting introduction to the breadth of the invention, theinvention includes several general and useful aspects, including:

1) Instrumentation including electrodes, and electrode arrays forreliably generating uniform electrical fields in cultures of livingcells in aqueous solution.

2) Multiwell plates comprising surface electrodes for high throughputand miniaturized stimulation and analysis of ion channel or cellularactivities.

3) Systems for high throughput analysis of ion channel and cellularactivities and for use in drug discovery, analysis, screening andprofiling.

4) Methods for modulating the transmembrane potential of a living cellvia the use of repetitive electrical stimulation.

5) Methods for screening the effects of test compounds on the activitiesof voltage regulated, and non-voltage regulated ion channels,transporters and leak currents. Including determining state-dependentpharmacological activity of compounds against ion channel andtransporter proteins.

6) Methods for profiling and selecting cells or clones based on theirresponse to electrical stimulation.

7) Methods for quantitative determination of cellular and ion channelparameters in a high-throughput manner, and for quantification of thepharmacological effects of compounds on those parameters.

8) Methods for the introduction of exogenous compounds into theintracellular spaces of cells.

9) Methods for modulating the transmembrane potential of intracellularorganelles, and for screening test compounds against ion channels inthese organelles.

10) Methods for characterizing the physiological effect of thetransmembrane potential on the function and regulation of physiologicaland biochemical responses, including gene expression, enzyme function,protein activity and ligand binding.

11) Methods for programming or training adaptive neuronal networks orbio-computers for specific functional or logical responses.

12) Methods for providing efficient neuronal interfaces for prostheticdevices implanted into an animal, including a human.

These aspects of the invention and others described herein, can beachieved by using the methods and instrumentation described herein. Togain a full appreciation of the scope of the invention, it will befurther recognized that various aspects of the invention can be combinedto make desirable embodiments of the invention. Such combinations resultin particularly useful and robust embodiments of the invention.

2. II Electrodes and Electrode Arrays

In one embodiment, the present invention includes electrodes, andelectrode arrays, for creating electrical fields across the area ofobservation. Typically this is achieved via the use of a pair ofelectrically conductive electrodes. An important design feature is thatthe electrode pairs create well-defined electrical fields. Preferredelectrode designs include electrode configurations that maximize theelectric field homogeneity experienced by the cells under observation.

Generating uniform electric fields over the area of observation isimportant for electrical stimulation for several reasons. Firstly,because the cellular response is sensitive to the magnitude of the localelectric field, non-uniform fields typically cause non-uniform responsesin different areas, leading to an increased scatter in the results.Secondly, the threshold for electropermeablization is typically only afactor of 2-5 larger than the transmembrane potentials required forelectrical stimulation membrane (see Teissie and Rols, 1993, Biophys. J.65:409-413). Thus, if the electric field is too non-uniform, it may notbe possible to stimulate all the cells in the area of observationwithout also electropermeablizing some of them.

Field uniformity over a fixed area can be described in two ways: (1) thestandard deviation of the field magnitude divided by the average fieldmagnitude in the area, and (2) the difference between the highest andlowest field magnitudes, normalized to the average field magnitude inthe area.

a. a) Design of Electrodes

The simplest way to generate a uniform electric field in a conductivemedium is to use two identical, flat electrodes with surfaces that arealigned substantially parallel to each other. Generally the closer theelectrodes are to each other relative to their width in the transversedirection, the greater the field uniformity will be. Typical roundmultiwell plate wells however limit the width of electrodes that can beinserted into the wells, and also introduce two other effects whichreduce field uniformity.

The roundness of the wells provides a challenge to create a uniformfield pointing in one direction with two electrodes the width of theconductive saline between the electrodes is constantly changing.Additionally the high surface tension of water generates variations inthe height of the saline across the well when dipper electrodes areinserted. The curved surface, or meniscus, can perturb the electricfield throughout the volume of the well. The depth of 100 μL of salinein a 96-well plate is normally about 3.0 mm deep at the center and about2.9 mm deep at the edges of the well. When two stainless steel parallelplate electrodes are inserted, saline is drawn up between the electrodesand the walls of the well causing depth variations over the area ofobservation suggesting that the current paths throughout the volume ofthe saline curve around the center, generating electric fieldnon-uniformity.

In one aspect the present invention includes improved electrode designs,and systems for electrical stimulation that address these issues tocreate substantially uniform electrical fields over the area ofobservation.

In one embodiment, (FIG. 9A) the electrode pair comprises twosubstantially parallel electrodes comprising an electrical insulatorthat is attached to the pair of electrodes to restrict current flow to adefined region thereby creating a highly uniform electrical field.

In another embodiment, (FIG. 9B) the electrode pair additionallycomprises satellite electrodes to create a more uniform electricalfield.

In another embodiment, (FIG. 9D) the electrode pair is sub-divided intoseveral pieces separated by thin insulating dividers. In this case thepotential applied to each electrode, expressed as a fraction of thepotential applied to the central most piece can be individually tuned tomaximize the field uniformity in the area of observation.

In another aspect, the present invention includes improved electrodedesigns (FIG. 9C) that exhibit improved field uniformity over the areaof observation via the elimination or reduction of the meniscus effect.

In another aspect multiple electric potential sensors can be fabricatedinto the surface or walls of the wells in a multiwell plate, or attachedin arrays to the dipper electrode assembly. These sensors can bemonitored to manually or automatically adjust the individual electrodes,so as to maximize field uniformity. This arrangement will be useful toallow a stimulating electrode array to compensate for variations andimperfections in the well shape, volume of saline, variations in themanufacturing process for the electrodes, damage to the electrodeassembly, etc.

b. b) Placement of electrodes within the wells

For dipper electrodes, the ideal situation (in terms of creating auniform electric field) would be to have the bottoms of the electrodestouching the bottom of the well. This way, there will be no fringingfields or field non-uniformity associated with vertical current paths.For a removable structure, however, it is not desirable to require theelectrodes to make contact with the surface. Small deviations in theplate geometry can cause some electrodes to press into the surface,causing damage either to the plate, the cells, or the electrodes.Additionally, in some wells, the electrodes may not extend all the wayto the surface. For these reasons it may be desirable to design a smallgap between the bottom of the electrode and the bottom of the well.

Accordingly in one aspect the present invention includes multiwellplates in which the area of observation in the middle of the well israised relative to area around the circumference of the well, where theelectrodes would be placed.

The fringing fields will cause non-uniformity over an inter-electrodedistance roughly equal to the gap between the bottom of the electrodeand the bottom of the well. Therefore, this gap should be kept as smallas is practical, preferably in the range of 0.1 to 0.5 mm and the areaof observation should not typically include any part of the well withinthis distance from the electrodes.

c. c) Manufacture of Electrodes

Any electrically conductive material can be used as an electrode.Preferred electrode materials have many of the following properties, (1)they do not corrode in saline, (2) they do not produce or release toxicions, (3) they are flexible and strong, (4) they are relativelyinexpensive to fabricate, (5) they are non porous, and (6) they areeasily cleaned. Preferred materials include noble metals (includinggold, platinum, and palladium), refractory metals (including titanium,tungsten, molybdenum, and iridium), corrosion-resistant alloys(including stainless steel) and carbon or other organic conductors(including graphite and polypyrrole). For many embodiments stainlesssteel provides a preferred electrode material. This material isinexpensive, easy to machine, and very inert in saline. Stainless steeloxidizes slowly to produce iron oxide when passing current in saline,but this does not appear to affect the performance of the system. Ironoxide has very low solubility in water and toxic levels of iron do notappear to be released. Additionally any iron oxide deposits can easilybe removed by soaking the electrodes in 10% nitric acid in water for twohours, then rinsing thoroughly with distilled water.

Solid copper and silver electrodes may be used for some applications butare less preferred for routine use because they corrode rapidly insaline. Gold plated copper electrodes are relatively inert, but appearto lose their gold plating during prolonged electrical stimulation.

Electrolysis products can be contained or eliminated by coating thesurfaces of the electrodes with protective coatings, such as gelatin,polyacrilimide, or agarose gels. Another potentially useful electrodematerial is an electrochemical half-cell, such as a silver/silverchloride electrode.

d. d) Electrode Arrays

Dipper electrodes typically consist of one or more pairs of electrodesthat are arranged in an array that can be retractably moved into, andout of, one or more wells of a multiwell plate. Dipper electrodes may beorientated into arrays that match the plate format and density, but canbe in arrays of any configuration or orientation. For example for astandard 96 well plate, a number of electrode configurations arepossible including electrode array arrangements to selectively exciteone or more columns, or rows, simultaneously.

An example of one embodiment of an electrode array of this type is shownin FIG. 1. In this example, a 12 by 8 array of electrode pairs isformatted so as to fit into a standard 96-well multiwell plate. In thiscase the electrodes (10) are approximately 4 mm wide, 1 cm long and 0.2mm thick, and extend from a conductive comb (50) that is connectedthrough a switch to one side of the output stage of a high-powerfunction generator. The electrodes are mounted parallel to each other, 4mm apart, with a non-conductive nylon spacer (20) in between. In thiscase, the switch (330) enables one column of the 96 well plate to beselectively stimulated at a time, however any temporal, or spatial,combination of stimulation protocols is potentially possible given theappropriate configuration of switching, wiring and power functiongenerator.

The entire array of electrodes is held in correct registration by arigid non conductive member (30) that keeps each electrode paircorrectly spaced to accurately match a standard 96 well plate layout.The non-conductive member (30) provides for the electrodes to move up ordown while precisely maintaining their registration with the multiwellplate.

To provide for correct registration of the electrode array with amultiwell plate, the electrode assembly can optionally comprise an outerborder or flange (40) that can accommodate a standard 96-well plate, andenables accurate plate registration. In some embodiments the border (40)can further include a registration notch or indentation (80) to provideunambiguous plate registration.

In a preferred embodiment (Also shown in FIG. 1A) the electrode arrayfurther comprises means for retractably inserting the electrode arrayinto the wells of the multiwell plate. In one embodiment of thisconfiguration, the electrode array further comprises an upper, movablesupport member (90) to which the electrodes (10) are attached. Themovable support member (90) is able to move up or down relative to thenon-conductive member (30) by sliding on four alignment pins (70). Notshown in these figures is a spring that enables the movable supportlayer (90) to automatically return to the upper position when downwardforce is no longer applied. A spacer (60) provides the ability to lockthe movable support layer (90) and electrodes (10) in the fully lowerorientation. This device allows the electrical stimulator to be used inmanual and/or robotic screening modes.

3. III Multiwell Plates for Electrical Stimulation

The multiwell plates of the present invention are designed primarily toprovide for efficient electrical stimulation of cells while at the sametime enabling the optical analysis of transmembrane potential changes.To accomplish this conductive surface electrodes may be orientated in,or on, the walls, bottoms or lids of the multiwell plate. In generalsuch multiwell plates can have a footprint of any shape or size, such assquare, rectangular, circular, oblong, triangular, kidney, or othergeometric or non-geometric shape. The footprint can have a shape that issubstantially similar to the footprint of existing multiwell plates,such as the standard 96-well microtiter plate, whose footprint isapproximately 85.5 mm in width by 127.75 mm in length, or other sizesthat represent a current or future industry standard (see T. Astle,Standards in Robotics and Instrumentation, J. of Biomolecular Screening,Vol. 1 pages 163-168,1996). Multiwell plates of the present inventionhaving this footprint can be compatible with robotics andinstrumentation, such as multiwell plate translocators and readers asthey are known in the art.

Typically, wells will be arranged in two-dimensional linear arrays onthe multiwell plate. However, the wells can be provided in any type ofarray, such as geometric or non-geometric arrays. The multiwell platecan comprise any number of wells. Larger numbers of wells or increasedwell density can also be easily accommodated using the methods of theclaimed invention. Commonly used numbers of wells include 6, 12, 96,384, 1536, 3456, and 9600.

Well volumes typically can vary depending on well depth and crosssectional area. Preferably, the well volume is between about 0.1microliters and 500 microliters. Wells can be made in any crosssectional shape (in plan view) including, square, round, hexagonal,other geometric or non-geometric shapes, and combinations (intra-welland inter-well) thereof. Preferred are square or round wells, with flatbottoms.

The walls can be chamfered (e.g. having a draft angle). Preferably, theangle is between about 1 and 10 degrees, more preferably between about 2and 8 degrees, and most preferable between about 3 and 5 degrees.

The wells can be placed in a configuration so that the well center-towell-center distance can be between about 0.5 millimeters and about 100millimeters. The wells can be placed in any configuration, such as alinear-linear array, or geometric patterns, such as hexagonal patterns.The well-to-well distance can be about 9 mm for a 96 well plate. Smallerwell-center to well-center distances are preferred for smaller volumes.

The wells can have a depth between about 0.5 and 100 millimeters.Preferably, the well depth is between about 1 millimeter and 100millimeters, more preferably between about 2 millimeters and 50millimeters, and most preferably between about 3 millimeters and 20millimeters.

The wells can have a diameter (when the wells are circular) or maximaldiagonal distance (when the wells are not circular) between about 0.2and 100 millimeters. Preferably, the well diameter is between about 0.5and 100 millimeters, more preferably between about 1 and 50 millimeters,and most preferably, between about 2 and 20 millimeters.

The multiwell plate, will generally be composed of electricallynon-conductive material and can comprise an optically opaque materialthat can interfere with the transmission of radiation, such as light,through the wall of a well or bottom of a well. Such optically opaquematerials can reduce the background associated with optical detectionmethods. Optically opaque materials can be any known in the art or laterdeveloped, such as dyes, pigments or carbon black. The optically opaquematerial can prevent radiation from passing from one well to another, toprevent cross-talk between wells, so that the sensitivity and accuracyof the assay is increased. The optically opaque material can also bereflective, such as those known in the art, such as thin metal layers,mirror coatings, or mirror polish. Optically opaque materials can becoated onto any surface of the multiwell plate, or be an integral partof the plate or bottom as they are manufactured. Optically opaquematerial can prevent the transmittance of between about 100% to about50% of incident light, preferably between about 80% and greater than95%, more preferably greater than 99%.

Since most measurements will not typically require light to pass throughthe wall of the well, materials such as polymers can include pigments todarken well walls or absorb light. Such application of pigments willhelp reduce background fluorescence. Pigments can be introduced by anymeans known in the art, such as coating or mixing during the manufactureof the material or multiwell plate. Pigment selection can be based on amixture of pigments to dampen all background inherent to the polymer, ora single pigment or ensemble of pigments selected to filter or absorblight at desired wavelengths. Pigments can include carbon black.

Surface electrodes can be embedded or otherwise attached to the wall ina variety of formats and arrangements, for example as several narrowvertical electrode stripes. By appropriately tuning the relativepotentials of each stripe, uniform electric fields can be generated inthe area of observation. Further, using a circular insert, or byembedding vertical stripe electrodes all around the well, uniformelectrical fields can be generated in any direction across the well. Itwould also be possible to create a uniform field in one direction,followed by a uniform field in another direction. This could be usefulfor cell types whose electrical characteristics are anisotropic, such asneural or muscle cells, or for cell types with large aspect ratios.

Each well also comprises a bottom having a high transmittance portionand having less fluorescence than a polystyrene-bottom of at least about90 percent of said bottom's thickness. This property can be determinedby comparing the fluorescence of an appropriate control bottom materialwith the fluorescence of a test material. These procedures can beperformed using well known methods. Preferably, the bottom is a plate orfilm as these terms are known in the art. The thickness of the bottomcan vary depending on the overall properties required of the platebottom that may be dictated by a particular application. Such propertiesinclude the amount of intrinsic fluorescence, rigidity, breakingstrength, and manufacturing requirements relating to the material usedin the plate. Well bottom layers typically have a thickness betweenabout 10 micrometers and about 1000 micrometers. Preferably, the wellbottom has a thickness between about 10 micrometers and 450 micrometers,more preferably between about 15 micrometers and 300 micrometers, andmost preferably between about 20 micrometers and 100 micrometers.

The bottom of a well can have a high transmittance portion, typicallymeaning that either all or a portion of the bottom of a well cantransmit light. The bottom can have an optically opaque portion and ahigh transmittance portion of any shape, such as circular, square,rectangular, kidney shaped, polygonal, or other geometric ornon-geometric shape or combinations thereof.

Preferably, the bottom of the multiwell plate can be substantially flat,e.g. having a surface texture between about 0.001 mm and 2 mm,preferably between about 0.01 mm and 0.1 mm (see, Surface Roughness,Waviness, and Lay, Am. Soc. of Mech. Eng. #ANSI ASME B46.1-2985 (1986)).If the bottom is not substantially flat, then the optical quality of thebottom and wells can decrease because of altered optical and physicalproperties of one or both.

For surface electrode embodiments, the bottom will preferably comprisestrips of electrically conductive material or coatings that overlap theedge of the wells of the multiwell plate and are in electrical contactwith the contents of the wells. The electrically conductive strips willtypically terminate at electrical connectors to enable facile attachmentto the output stage of a high-power function generator as describedpreviously. The electrically conductive strips should have low enoughresistance so that they can carry the stimulating currents withoutexcessive loss in voltage over their length. The resistance from theconnector end to the farthest well end should be less than 10 Ω, andmore preferably less than 1 Ω, and more preferably still less than 0.1Ω. The cross-sectional area of the electrically conductive strips shouldbe large enough to accomplish the resistance requirement. For commonlyemployed electrical conductors, this cross sectional area should be atleast 10⁻⁴ mm², and more preferable at least 10⁻³ mm².

In practice, any conductive materials could be used as long as they arecapped with a conductive material that is inert in saline. Suchmaterials include the noble metals (including gold, platinum, andpalladium) and the refractory metals (including chromium, molybdenum,iridium, tungsten, tantalum, and titanium) as well as alloys thereof.Preferred materials for the conductive material for surface electrodesinclude combinations of chromium, copper, gold, and indium-tin-oxidethat can be readily embedded or electroplated into or on the transparentbottom layer. Electrolysis products can be contained or eliminated bycoating the surfaces of the electrodes with protective coatings, such asgelatin, polyacrilimide, or agarose gels.

Another potentially useful electrode material is an electrochemicalhalf-cell, such as a silver/silver chloride electrode.

The electrically conductive material coatings or surface modificationscan be introduced into the bottom using any suitable method known in theart, including vacuum deposition, electroplating, printing, spraying,radiant energy, ionization techniques or dipping. Surface modificationscan also be introduced by appropriately derivatizing a polymer or othermaterial, such as glass or quartz, before, during, or after themultiwell plate is manufactured and by including an appropriatederivatized polymer or other material in the bottom layer. Thederivatized polymer or other material can then be reacted with achemical moiety that is used in an application of the plate. Prior toreaction with a chemical moiety, such polymer or other material can thenprovide either covalent or non-covalent attachment sites on the polymeror other material. Such sites in or on the polymer or other materialsurface can be used to attach conductive layers to the plates. Examplesof derivatized polymers or other materials include those described byU.S. Pat. No. 5,583,211 (Coassin et al.) and others known in the art orlater developed.

(i) Materials and Manufacturing

The materials for manufacturing the multiwell plate will typically bepolymeric, since these materials lend themselves to mass manufacturingtechniques. However, other materials can be used to make the bottom ofthe multiwell plate, such as glass or quartz. The bottom can be made ofthe same or different materials and the bottom can comprise polystyrene,or another material. Preferably, polymers are selected that have lowfluorescence and or high transmittance. Polymeric materials canparticularly facilitate plate manufacture by molding methods known inthe art and developed in the future, such as insert or injectionmolding.

The multiwell plate of the present invention can be made of one or morepieces. For example, the plate and bottom can be made as one discretepiece. Alternatively, the plate can be one discrete piece, and thebottom can be a second discrete piece, which are combined to form amultiwell plate. In this instance, the plate and bottom can be attachedto each other by sealing means, such as adhesives, sonic welding, heatwelding, melting, insert injection molding or other means known in theart or later developed. The plate and bottom can be made of the same ordifferent material. For example, the plate can be made of a polymer, andthe bottom made of polystyrene, cycloolefin, Aclar, glass, or quartz.

Miniaturized surface electrode designs are feasible in standard plateformats (96, 384, 1536) as well as 3456 and higher plate densities. Thethroughput of such systems is potentially extremely high. For example,assuming 3456 wells per plate screened at 30 plates per hour correspondsto an overall throughput of approximately 800,000 wells per eight-hourday, which is approximately 8 times faster than is presently available,assuming equal plate read times.

An example of one embodiment of multiwell plate with surface electrodesis shown in FIG. 2A. In this example, pairs of conductive strips (200)are attached in parallel to an optically transparent bottom layer (210)such as glass, or plastic such as COC (see U.S. Pat. No. 5,910,287,issued Jun. 8, 1999) in a 96-well plate format. In this example, thestrips of conductive material (200) are approximately 2 mm wide, 10 μmthick, and separated by distance of approximately 4 mm to enable opticalanalysis of the cells located in the wells (220), between the electrodesthrough the optically transparent bottom layer (210). In otherembodiments the strips of conductive material can comprise stainlesssteel wires (from about 0.001″ to about 0.010″ diameter). The opticallytransparent bottom layer (210) is attached to a 96-well multiwell platearray (230) and replaces the normal plate bottom. The strips ofelectrically conductive material (200) overlap the edge of the wells(220) of the 96-well multiwell plate and are in electrical contact withthe contents of the wells. The electrically conductive strips (200)terminate at electrical contacts (240) to enable facile attachment tothe output stage of a high-power function generator as describedpreviously. In this example, there are two electrode contacts pereight-well column in the first well of the column. This permits the useof standard 96-well plate layouts, for simpler handling during cellculturing. No cells or saline are inserted into these wells. This designpermits the simultaneous stimulation of seven wells in a single column.During the assay, the operator or a robot will temporarily attach wiresto the contacts, for example with push-pin test electrodes.

Another embodiment of a multwell plate with surface electrodes is shownin FIG. 2 b. In this embodiment, the transparent bottom layer (210)extends beyond the edge of the multiwell plate (230). In thisconfiguration, all wells remain available for use with cells andcompounds. Further, attachment of external wiring to the contacts (240)is simplified. Push-pin contacts, circuit-board edge connectors, orzero-insertion force sockets can be used to make contact with theelectrodes. The extended bottom layer (210) may make the plates lessconvenient to manipulate during routine use. This can be remedied bybringing the electrode traces (200) to the reverse side of the bottomlayer (210) during the manufacturing process. This can be accomplishedby several methods. For example, using two-sided processing of theplates to create contact areas, through-holes can be made andelectroplated, or conducting traces can be wrapped around the edge ofthe bottom layer. As another example, the bottom layer can be made of aflexible insulating material. Then, after making the structure as shownin FIG. 2B, the part of the bottom layer which protrudes from the edgeof the plate can be folded and attached to the underside of the plate.

Another embodiment of a multwell plate with surface electrodes is shownin FIG. 2C. In this embodiment, the electrodes (200) are attached to thecontact pads (240) with narrow via wires (205). This permits the use ofstandard 96-well plate layouts, for simpler handling during cellculturing. In this embodiment, all of the electrodes of one polarity areshorted together. Selection of a single column is accomplished bysupplying the current pulse to only one electrode of the other polarity.In this embodiment, no cells, saline, or compounds are placed into thefinal column where the contact pads are. During the assay, the operatoror a robot will temporarily attach wires to the contacts, for examplewith push-pin test electrodes.

Another embodiment of a multwell plate with surface electrodes is shownin FIG. 2D. In this embodiment, the electrodes (200) are alignedparallel to the longer dimension of the 96-well plate. This design isessentially similar to the design shown in FIG. 2A, with the exceptionthat eleven wells in a row will be simultaneously stimulated.

Preferred materials for the conductive material for surface electrodesinclude combinations of chromium, copper, gold, and indium-tin-oxidethat can be readily embedded, attached, or electroplated into or on thetransparent bottom layer. In practice, any conductive materials could beused as long as they are capped with a conductive material that is inertin saline. Such inert materials include the noble metals (includinggold, platinum, and palladium), the refractory metals (includingchromium, molybdenum, iridium, tungsten, tantalum, and titanium),corrosion-resistant alloys (including stainless steel), and carbon orother organic conductors (including graphite and polypyrrole) as well ascombinations or alloys of these materials.

4. IV Systems for Electrical Stimulation and Spectroscopic Measurement

The present invention includes systems for automated electricalstimulation and spectroscopic measurement, comprising: at least oneelectrode assembly, a means for electrical stimulation, an opticaldetector, and computer control means to coordinate the generation ofelectrical stimuli, collection of data and movement of multiwell plates.The system can further comprise means for fluid addition. In one aspectthese systems are designed for modulating, characterizing and assayingthe activity of ion channels, transporters, leak currents present in oron the surfaces of living cells, and for rapidly screening for theeffects of test compounds on the effects of channel or cellularactivities. The present invention is also directed to chemical entitiesand information (e.g., modulators or chemical or biological activitiesof chemicals) generated or discovered by operation of workstations ofthe present invention.

FIG. 3 shows a block diagram of the major electrical and opticalcomponents for one embodiment of a system for automated electricalstimulation and spectroscopic measurement. In this example a 96-wellmultiwell plate dipper electrode array (FIG. 1) was used for electricalstimulation. In addition to the stimulator electrode array, the systemhas several additional electrical, optical and mechanical components, asdescribed in detail in commonly owned U.S. patent application Ser. No.09/118,728, filed Jul. 24, 1998.

In this embodiment, a National Instruments (Austin, Tex.) PC-DIO 24digital input/output card on board the computer (300) is used to set theproper channel on a 1-to-12 switch (330) (National Instruments ER-16).The computer controlling the fluorescent plate reader (300) also sendsout a TTL signal to trigger the function generators (310) when thestimulus is programmed to begin. Stimulus signals are generated by twoarbitrary waveform generators (310). The function generators areTektronix (Beaverton, Oreg.) model number AFG310. The first triggers aseries of TTL pulses to the second which is programmed with theindividual stimulus waveform. More complex waveform trains can begenerated by connecting multiple waveform generators in series and/or inparallel. These waveform generators would be triggered by thecomputer-generated TTL pulse or by each other. Alternatively, an A/Dconverter or a sound card on board the computer could be used togenerate a train of stimuli. In this case, commercially-available orcustom software could be used to program the waveform train, or tochange the waveform during the train.

The train of stimuli is sent through a high-power amplifier (320),through the switch (330), and into the stimulator head (370). In thiscase the amplifier was built using the APEX PA93 chip (ApexMicrotechnology Corp, Tucson, Ariz.) following a circuit provided by themanufacturer. Preferred amplifiers for the present application wouldtypically meet, or exceed the following specifications: ±100V DC in, 100GΩ input impedance, 20×voltage gain, ±90V out, ±3 A out, 10 Ω outputimpedance.

The majority of current passes through the saline between theelectrodes, typically in a single eight-well column of the microtiterplate (350) at a time. Excitation light at 400±7.5 nm illuminates thestained cells from below, and emitted fluorescent light is measured attwo wavelengths via the detector module (340) blue at 460+/−20 nm andorange at 580+/−30 nm; (see González et al., Drug Discovery Today 4:431-439, 1999). Once a column of cells has been stimulated the computer(300) triggers the motor (360) to move the multiwell plate (350) to anew position ready for the next stimulation.

For a typical 96-well multiwell plate, the electrodes are 4 mm wide witha gap (g) of 4 mm. Stimulation is usually performed in a volume of 100μL of physiological saline in the well. With this volume of saline, thedepth averages approximately 3.0 mm (this depth varies by as much as 20%across the well due to the meniscus effect). The electrodes restapproximately 0.5 mm off the bottom of the wells. The electric field (E)applied across the cells is estimated as the voltage across theelectrodes (V₀) divided by the electrode gap (g), E=V₀/g . This is anoverestimate of the actual field because of the influence ofelectrochemical reactions at each electrode which consume approximately1.5 V. In the typical voltage ranges used for stimulation (10 to 60V/cm), this overestimate is on the order of approximately 10%. Accuratemeasurement and calibration of the field can be performed by mapping theelectric potential in the well when current is passed.

The present invention also includes automated workstations that areprogrammably controlled to minimize processing times at each workstationand that can be integrated to minimize the processing time of the liquidsamples for electrical stimulation and analysis.

Typically, a system of the present invention would include one or moreof the following: A) a storage and retrieval module comprising storagelocations for storing a plurality of chemicals in solution inaddressable chemical wells, a chemical well retriever and havingprogrammable selection and retrieval of the addressable chemical wellsand having a storage capacity for at least 100,000 addressable wells, B)a sample distribution module comprising a liquid handler to aspirate ordispense solutions from selected addressable chemical wells, thechemical distribution module having programmable selection of, andaspiration from, the selected addressable chemical wells andprogrammable dispensation into selected addressable sample wells(including dispensation into arrays of addressable wells with differentdensities of addressable wells per centimeter squared), C) a sampletransporter to transport the selected addressable chemical wells to thesample distribution module and optionally having programmable control oftransport of the selected addressable chemical wells (including adaptiverouting and parallel processing), D) a system for automated washing,staining, and timed incubation of cells in multiwell plates, E) a systemfor automatically transporting cell plates and test compound platesbetween the various workstations, F) a system for automated electricalstimulation and spectroscopic measurement, and a data processing andintegration module, G) a master control system which co-ordinates theactivities of any of the above subsystems.

The storage and retrieval module, the sample distribution module, andthe system for automated electrical stimulation and spectroscopicmeasurement are integrated and programmably controlled by the dataprocessing and integration module. The storage and retrieval module, thesample distribution module, the sample transporter, the system forautomated electrical stimulation and spectroscopic measurement and thedata processing and integration module are operably linked to facilitaterapid processing of the addressable sample wells. Typically, devices ofthe invention can process at least 100,000 addressable wells in 24hours. This type of system is described in U.S. Pat. No. 5,985,214,issued Nov. 16, 1999, which is incorporated herein by reference.

a. d) Microfluidic Systems

The present invention also includes the use of electrodes that have beenincorporated into microfluidic chips and which provide for highlyminiaturized electrical stimulation and analysis. Such systems includethose, for example, described in U.S. Pat. No., 5,800,690 issued Sep. 1,1998 to Chow et al., European patent application EP 0 810 438 A2 filedMay 5, 1997, by Pelc et al. and PCT application WO 98/00231 filed 24Jun. 1997 by Parce et al. These systems typically use electrogenic fluidmovement to manipulate small fluid volumes within microcapillariespresent on glass or silicon chips. These microfluidic chip basedanalysis systems can provide massively parallel miniaturized analysis.Such systems are preferred systems of spectroscopic measurements in someinstances that require miniaturized analysis.

For example, the microfabricated fluorescence-activated cell sorterdescribed by Fu et al. (Nature Biotechnology 17: 1109-11, 1999) could beeasily modified to have a pair of electrodes placed in, or near theoptical interrogation region. Using the methods described herein,individual cells could be electrically stimulated and individuallysorted based on their response to the stimulation. This method wouldgreatly simplify the process of obtaining stable clones containing thedesired expression of channels. In another aspect, screening of testcompounds on single cells could be performed with a microfluidic deviceequipped with one or more additional fluid injection ports and one ormore embedded electrical stimulator devices built and operated based onthe methods described herein.

5. V Electrical Stimulation Methods

a. a) Introduction

Without being bound to any mechanism of action, the present inventorsprovide the following description for the effect of electricalstimulation on cellular transmembrane potentials.

Typical voltage-dependent ion channels have a variety of conducting andnon-conducting states that are regulated by the local relativetransmembrane potential of the cell. By appropriately applying externalelectrical fields to the cells, portions of the cell membrane can bedriven to any desired transmembrane potential, thereby enabling theregulation of the conduction states of voltage dependent ion channelspresent within the cell. If the applied electrical field isappropriately varied, it is possible to sample a number of conductancestates of most ion channels, thereby cycling them through resting,activated, and inactivated states.

Depending on the ion channel in question, activation of the ion channelcan lead to the release, or uptake, of ions into the cell that canresult in global transmembrane potential changes in the cell. Byapplying a repetitive train of electrical stimuli, separated by a timeinterval smaller than the membrane time constant, large sustainedmembrane voltage changes can be created via a stepwise accumulation orloss of ions. This process allows the direct measurement of many ionchannels and provides a facile method whereby the transmembranepotential of the cell can be externally controlled. This approachtherefore provides for improved methods of drug discovery that arecompatible with high throughput screening.

b. b) Overview of a Typical Stimulation Protocol

The simulated influence of a typical biphasic electrical stimulationprotocol on a cell line expressing a voltage activated sodium channel isillustrated, in simplified form, below. The following descriptionassumes that the cell line has no significant expression of other ionchannels, and that the resting transmembrane potential of the cell isabove the threshold for inactivation of the sodium channel in question.In FIG. 4, the upper panel shows the time course of the appliedelectrical field (E), the middle panel shows the simulated inward sodiumcurrents (I_(Na)) in response to the applied electrical field, and thelower panel shows the idealized average transmembrane potential of thecell (V_(m)). In this example, the recordings relate to the changes inthese parameters that a single cell placed in the center of the appliedelectrical field would be typically expected to experience during anelectrical stimulation wave train.

Referring to the first pulse, establishing the first electrical fieldcauses a potential drop across the cell that is maximal, with respect tothe resting transmembrane potential of the cell, at the edges of thecell closest to the electrodes (see Hibino et al., Biophysical Journal64:1789-1800, 1993; Gross et al. 1986, Biophys. J. 50:339-348). Themagnitude of the electric field-induced transmembrane potential changeΔ_(Vm) at a given point of the membrane in an idealized spherical cellcan be described by the formula (Ehrenberg et al., Biophys. J.51:833-837, 1987):

$\begin{matrix}{{\Delta\; V_{m}} = {{- f}\; g\; r\; E\mspace{11mu}\cos\;{\theta.}}} & (1)\end{matrix}$

In Equation 1, f is a factor dependent upon the conductivity of themembrane, g is a geometric factor of order 1, r is half the diameter ofthe cell parallel to the electric field, E is the local magnitude of theelectric field, and θ is the angle between the local direction of thefield and a line drawn from the center of the cell to the point of thesurface being considered. For most intact mammalian cells, in which themembrane conductivity is very low compared to the conductivity of thesolution bathing the cells, the factor f≈1. In practice, cells arerarely spherical when attached to a substrate and an accurate estimateof the actual magnitude of the electrical field induced transmembranepotential changes may be empirically determined.

As a result of the applied electrical field, the membrane on the sidenearest to the anode is driven negative, while the membrane on the sidenearest the cathode is driven positive. In cells in which one edge isdriven sufficiently negative to locally lower the transmembranepotential below the threshold potential for release of inactivation forthe ion channel in question, the applied electrical field causes thesodium channels located on this edge to enter the resting state. On theother side of the cell, the transmembrane potential is driven positiveof the resting potential. Because the resting transmembrane potential ofthe cell is assumed to be above the threshold for inactivation, sodiumchannels on this side of the cell remain inactivated and do not passcurrent. If the resting transmembrane potential were instead below theinactivation threshold, channels on this side of the cell would activateand pass current.

When the applied field is reversed, the profile of transmembranepotential changes also reverses. The transmembrane potential changesinduced by the electric field on the patches of membrane at the extremeedges of the cells switches polarity. The channels on the side that wasdriven negative during the first phase of stimulation are now drivenpositive. If the stimulation parameters are properly chosen, thesechannels are now driven above the activation potential and begin toallow sodium ion influx. This is shown in FIG. 4, as the first smallerpeak of sodium influx into the cell. The sodium channels rapidlyinactivate after a characteristic time. Meanwhile, on the other side ofthe cell, the transmembrane potential is driven negative so that thesodium channels release from inactivation and move into the restingstate.

When the second stimulus phase ends, all parts of the membrane rapidlyreturn to a new average transmembrane potential. If the averagetransmembrane potential is now above the activation potential of thesodium channels, the channels on the side of the cell that was drivennegative during the second phase of stimulation activate and begin toallow sodium ion influx. This is shown in FIG. 4, as the second largerpeak of sodium influx into the cell. The sodium channels rapidlyinactivate after a characteristic time. In this case sodium influx istypically larger from the second side than the first side, since thedriving force for sodium entry is larger when this part of the membraneis driven more positive by an electric field.

Each pulse of sodium channel influx raises the average transmembranepotential of the cell (FIG. 4, lower panel). This rise in transmembranepotential can be detected by any of the methods described herein, but isconveniently measured via fluorescence emission ratio changes of a FRETbased voltage-sensitive dye. Due to leakage currents present in allcells, this average transmembrane potential shift decays exponentiallyto the original resting transmembrane potential. The time dependency ofthis response, the membrane time constant (τ_(m)), depends upon themembrane capacitance and membrane resistance, and is highly variablefrom one cell type to another. For example, time constants can vary from100 μs to over one second, depending on the cell type. Typically themembrane time constant is around 100 ms for most engineered cell lines.To provide a net accumulation of sodium influx the stimulus pulse isrepeated before the transmembrane potential has time to decay to theresting transmembrane potential. During subsequent rounds of electricalstimulation, positive charge is steadily accumulated into the cellraising the average transmembrane potential in an approximately stepwisefashion with each repetition of electrical stimulation. After each pulseof electrical stimulation, the magnitude of the sodium ion influxesbecome steadily smaller as the average transmembrane potentialapproaches the sodium ion reversal potential. Eventually an equilibriumtransmembrane potential is established in which leakage of current outof the cell equals the current influx due to electrical stimulation.

c. c) Adjustable Parameters for the Stimulus Waveforms

The present invention includes the use of any waveform kernel with anyrepetition procedure capable of selectively activating ion channels inliving cells. The kernel is the repeatable structure that forms thebasis of the stimulus train. In FIG. 4, the kernel is a biphasic squarepulse, but in principle it could be any limited-time wave function. Thetime duration of the kernel sets the maximum rate at which it can berepeated. The repetition procedure dictates how and when the kernel ispresented to the sample. In FIG. 4, the repetition rate is fixed andcontinues for a total of ten cycles. However the repetition rate neednot be fixed.

Additionally, the kernel can be changed during the stimulus train, sothat each time the repetition procedure calls for a stimulus pulse, adifferent wave function could be used. Furthermore, a feedback mechanismcould be used to alter the kernel and/or the repetition procedure basedupon the measured response of the system.

The use of arbitrary waveform generators to create the stimulus kernelsand trains allows for a virtually unlimited variation in the waveform inorder to tune the electrical stimulus to a particular cell type orspecific ion channel. The pulse train can be readily modulated via thevariation of a number of separately controllable components.

(a) 1. The Shape of the Individual Pulses.

The waveform kernel that is repeated during the stimulus train can bechanged with nearly endless permutations using a arbitrary digitalwaveform generator, such as Tektronix AFG 310. FIG. 5 shows a schematicrepresentation of a biphasic square waveform to illustrate some of thevariables that can be modulated. In FIG. 5, the pulse train consists ofa starting field E₁ (400), that lasts for a time t₁, a rapid increase inpotential (410), that takes a time t₂, until reaching a firststimulating field E₂ (420) that lasts for time t₃, a rapid decrease inpotential (430) that takes time t₄, until reaching a second stimulatingfield (440), E₃ that lasts a time t₅, a rapid increase in potential(460) that takes time t₆, until reaching the finishing field (470), E₄that lasts a time t₇ until the cycle is repeated. The magnitude andpolarity of the electrical fields E₁ to E₄ are separately controllableand may be both statically and dynamically varied as described below.The times for which the electrical potentials are applied to the cells,times t₁, t₃, t₅, and t₇ are also separately controllable and may beboth statically and dynamically varied between 0 and 10 s during a wavetrain, as described below. Finally the changes in potential that occurover times t₂, t₄ and t₆, may occur over variable time periods between 0and 100 ms and be either linear or non linear to create waveforms ofvariable shapes.

Some examples of these types of variation in the waveform are shown inFIG. 6( a) Biphasic waveform, as shown in FIG. 5, repeated at a rate f.(b) A modified biphasic waveform. A short interval has been addedbetween the stimulation phases of the wave train. This allows current toflow through the channels released from inactivation during the firstpulse. (c) Monophasic waveform. Only channels on the side of the cellfacing the anode will be released from inactivation. (d) A rampedwaveform. The anode-facing channels will be released from inactivationby the square wave. The channels will activate and pass current duringthe ramp. The ramp allows the channels to open and pass current at morenegative local potentials, so that even when the cell is near thereversal potential for sodium ions, large currents can still flow. Thepoint along the ramp at which the channels will open varies. (e) Abiphasic triangular or sawtooth waveform. Ramping may allow thevoltage-dependent transitions between states to occur more uniformly asthe global membrane potential changes. Monophasic triangular waveformsare also possible. (f) A sinusoidal waveform. This type of waveform mayreduce electrical noise during high frequency stimulation. (g) A shortburst of sinusoidal waveforms. (h) Bursts of sinusoidal waveforms, eachwith different fundamental frequency. This type of stimulation may proveuseful for studying plasticity effects. The first burst(s) are used totrain the system or begin a process, while the subsequent bursts(s) areused to assay the system.

Variations in waveform shape may be useful in maintaining fixed stimulusconditions during the pulse train. For example, the transmembranepotential excursions experienced by a highly polarized cell will vary asits average transmembrane potential changes from around −90 mV at thebeginning of the stimulation cycle to around +60 mV after severalrepetitive stimulation cycles. As a consequence, the applied electricalfield required to efficiently release an ion channel from inactivationvaries as the average potential of the cell varies during the course ofseveral stimulation cycles. To take this effect into consideration itmay be useful, under certain circumstances, to change the relativebalance between the positive (E₂) and negative (E₃) phases ofstimulation as the wave-train progresses.

Some cell lines, for example HEK-293, have a resting averagetransmembrane potential below the activation threshold of somevoltage-activated sodium channels. In these cells as the transmembranepotential rises during stimulation as a result of sodium ion influx, thesodium channels can open independently of the applied electricalstimulation. This can be improved by using a sloped current pulse (i.eby increasing t₂ and t₄). Then, the channels can pass current for adefined time just above the activation voltage, independent of theaverage transmembrane potential of the cell.

(b) 2. The Overall Amplitude of the Individual Pulse (E₂ and E₃).

The magnitude and polarity of the pulse amplitude controls the relativetransmembrane potential excursions experienced by the cell during astimulus pulse. Pulse amplitudes can be altered for the entire train, orfor the individual pulses to accommodate different channels and celltypes, as discussed in more detail below. In general, the magnitudes ofE₂ and E₃ are selected to ensure that the ion channel of interest isefficiently activated, and released from inactivation during eachstimulation cycle, while at the same time not of sufficient magnitude soas to cause irreversible electroporation of the cells. Preferred pulseamplitudes for E₂ and E₃ are typically in the range of 5 to 60 V/cm formost ion channels when expressed in non-excitable mammalian cells withaverage sizes from 10 to 25 μm, and may vary either positive or negativerelative to earth. As above, the amplitude of the stimulus can bechanged during the pulse train to maintain stable stimulus conditions asthe average transmembrane potential changes. Preferred pulse amplitudesare inversely dependent upon average cell size. So, the technique canalso be used on cells which are smaller or larger than 10 to 25 μm, byaltering the pulse amplitude.

(c) 3. The Duration of the Individual Pulses (t₃ and t₅).

Many channels require alterations in the transmembrane potential forextended periods of time to release them from inactivation, prior toopening. For example, many voltage-dependent sodium channels generallyneed to experience a transmembrane potential below −90 mV for severalmilliseconds before they are released from inactivation. Efficient useof the electrical stimulation protocol therefore typically requires thatthe duration of the pulses t₃ and t₅ are sufficient to enable complete,or almost complete, release from inactivation for the ion channel ofinterest. In some cases it may be desirable to tune the magnitude of t₃and t₅ to enable the selective release from inactivation of one class,but not another class of ion channel in a cell that expresses severalion channel types. In other cases it may be desirable to make t₃ and t₅very small to achieve low levels of release from inactivation for thechannels. Typically the preferred pulse duration is matched to thecharacteristic time for transitions between the desiredvoltage-dependent states for the ion channel of interest, and these aretypically in the range of about 0.1 to 100 msec for most ion channels.

To avoid excessive electrolysis of water and consequent gas bubblegeneration, the duration of the pulses t₃ and t₅ should be kept as shortas possible, while still achieving the desired electrical stimulation.Water electrolysis at a metal/water interface typically occurs when themagnitude of the voltage difference between the metal and the waterexceeds about 0.8 V. In some cases, the stimulus parameters required toproduce cellular stimulation also cause water electrolysis. Somegeneration of gas at the electrodes is typically acceptable as long asthe charge per unit area of the electrode/water interface deliveredduring any single polarity phase of a single pulse is less than about100 μC/mm². Exceeding this limit typically causes gas evolution andbubble formation that significantly affects field uniformity. Thepresence of bubbles on the electrode surface occludes that part of theelectrode, and can cause alterations in the electric field uniformity.Generation of large amounts of gas can also cause oxidative damage tothe cells and the dyes in the well.

In a 96-well plate with 100 μL of physiological saline with resistivity70 Ω-cm, the resistance of the saline between two parallel plateelectrodes with a 4 mm gap between them inserted into the well to within0.5 mm of the bottom of the well, is approximately 230 Ω. Each electrodehas a contact area with the saline of about 24 mm². Thus, anysingle-polarity phase of the stimulus protocol should not deliver morethan about 2.4 mC of charge. A voltage difference of about 10 V appliedbetween the plates generates an electric field of about 25 V/cm in thesaline. This voltage will draw about 43 mA of current. Thus for thiselectrode configuration, a square wave, single-polarity pulse should notexceed about 55 milliseconds in duration in order to limit the charge toless than 2.4 mC.

(d) 4. The Gap Between Successive Stimuli (t₁ and t₇).

Changing the value of t₁ and t₇ globally for the train, or adjusting itfor each individual pulse during the train, is useful for optimizing thestimulation protocol for specific ion channels. Additionally theapproach is also useful for determining certain cellular and channelproperties including the open channel time and the time course of thechannel activation and inactivation.

For example, for assays involving voltage regulated sodium channels, theinsertion of a time delay (t₁+t₇) between pulses equal to, or less than,the average sodium channel open time allows for a quantitativemeasurement of the inactivation kinetics of the channel. Theinactivation kinetics are directly related to the average open channeltime. Thus, assays using short interpulse intervals allows for thedetection of compounds whose primary effect is on inactivation kinetics,a mechanism which is otherwise inaccessible using high-throughputtechniques.

In most cases the time delay between successive stimuli would be lessthat the membrane time constant in order to obtained sustained increasesin transmembrane potential. Typically optimal frequencies of stimulation(f) are within the range τ_(M) ⁻¹≦f≦τ_(b) ⁻¹ where τ_(M) is the timeconstant for decay of transmembrane potential changes, and τ_(b) is theaverage channel open time. Some channels do not inactivate, and forthese cells the stimulation frequency may be determined empirically.Additionally, the stimulation frequency f cannot exceed the inverse ofthe time duration of the stimulus kernel.

Additionally, for certain cell types, it may prove desirable tostimulate at a slower rate. For example, slower stimulation rates may bepreferred for cells with high channel densities, or for assays in whichhigher pharmacological sensitivity is required. Alternatively for thesecases, a monopolar stimulus could be used. This would only release frominactivation the sodium channels on one side of the cell, but themaximum frequency of stimulation could be doubled.

(e) 5. The Duration of the Train of Pulses, or Number of Pulses in theTrain.

Cellular and channel properties can be assayed both in dynamic (i.e.rise and fall times, alterations in response shape, etc.) and staticmodes. Both modes require stimulus train durations long enough toexplore all the events of interest, yet not longer than necessary tocomplete the assay. Typical stimulation times comprise 10 msec pulses,at 25 V/cm pulses repeated at a frequency of 20 Hz for 3 seconds.Adjusting these parameters allows assay times to be reduced, or toexplore processes with both fast and slow time scales.

(f) 6. Multiple Pulse Trains.

In some cases it is useful to repeat pulse trains, or to perform ameasurement on the same cells with two different pulse trains. Oneexample would be to completely characterize the properties of a channelby measuring the response as a function of stimulus frequency andduration, using a single plate of cells subjected to multiple stimulustrains. Another example would be to examine plasticity of the response(i.e. activity-dependent changes in response). One or more stimulustrains would condition the response, while sets of measurement trainsbefore and after the conditioning would determine the changes due toactivity.

(g) Feedback of Stimulus Parameters Based Upon Dynamic Measurements ofthe Response.

The present invention can also be used to create a voltage clamp device,by using a dynamic feedback loop to maintain the average transmembranepotential at a preset value. By measuring the transmembrane potentialusing a fast fluorescent output as described below, then changingstimulus parameters to compensate for any changes in transmembranepotential, it is possible to dynamically control the transmembranepotential of the cells. The current necessary to maintain that potentialwould then be determined by computer control of the stimulus parameters.

(h) The Use of High Frequency Stimulation to Avoid Electrolysis

During typical stimulation parameters, a peak current of approximately50 mA passes through the solution between the electrodes. During thistime various electrochemical reactions occur which typically generatetoxic species to the cells. Preliminary experiments have shown that mostmammalian cells typically respond normally for approximately two minutesof electrical stimulation using stainless steel electrodes. Howeverprolonged stimulation for longer time periods appears to lead to a lossin cell health and viability. At sufficiently high pulse frequencies,such that the metal-saline interface does not reach the potential forelectrolysis of water (approximately ±1V for stainless steel in saline),current can be passed capacitively and no toxic products will begenerated. In the electrical stimulator shown in FIG. 1, in which eachelectrode has an area of about 24 mm² in contact with the saline, thecapacitance per electrode is around 1-10 μF (Robinson, 1968, Proc. IEEE56:1065-1071). At 50 mA, this capacitance charges to 1 V in around20-200 μs. This is at the lower limit of the useful pulse durationtimes.

Alternatively it is possible to perform electrical stimulation withoutgenerating electrolytic products. Several treatments are available whichcan increase the capacitance of the metal-saline interface by factors of2-100. These include surface roughening, electroplating with platinumblack or gold black, and deposition and activation of iridium/iridiumoxide, titanium/titanium nitride, or polypyrrole films. Usingstimulation parameters, which avoid irreversible electrochemistry, thesesurface treatments do not degrade when passing current.

6. VI Expression of Ion Channels

a. a) Selection of the Cell Type

The present invention can be used with any type of cell, includinganimal cells, plant cells, insect cells, bacterial cells, yeast andmammalian cells. For screening for human therapeutics mammalian celllines are preferred, such cell lines include tissue culture cell linesthat can be relatively easily grown, and can be readily transfected withhigh efficiency. Many tissue cell lines are commercially availablethrough the American type culture collection (ATCC) see(http://www.atcc.org), as well as the European collection of cellcultures (ECACC) (http://www.camr.org.uk).

Additionally in some cases primary cell lines, or tissue slices may alsobe preferred for screening when it is required to express, or measure,the response of the ion channel of interest in its native physiologicalcontext. This approach may be useful either as a primary or a secondaryscreen to screen for specificity, selectivity or toxicity of candidatetherapeutics, and is discussed in detail in section X.

For assays performed on cultured cell lines, the main selection criteriaare the resting transmembrane potential of the cell line, and thepresence of endogenously expressed ion channels. The selection ofappropriate cell lines for specific ion channels of interest aredependent on the voltage dependent properties and ion selectivity of theion channel of interest. These considerations are reviewed in detail fora number of ion channels in section VIII, Stimulation Protocols.

In some cases it is desirable to use a cell line which has no (or verylow) detectable endogenous expression of other ion channels. Cells ofthis type include CHO-K1, CHL, and LTK(−) cells. These cells inherentlyhave a resting potential in the range of +10 to −30 mV, which is abovethe activation and inactivation thresholds of most voltage-dependentchannels. Use of these cell lines has the advantage that the ion channelof interest is the major modulator of transmembrane potential within thecells so that screening assay data are generally easily andunambiguously interpreted.

In some cases the use of a cell line with no other ion channels may notbe practical to create a workable assay. For example, it may benecessary to maintain a voltage-regulated ion at a highly polarizedtransmembrane potential. In this case it is necessary control thetransmembrane potential via the expression of a second ion channel. Forexample to assay a rat brain type IIa sodium channel in the restingstate requires the transmembrane potential to be maintained below thethreshold activation potential of the sodium channel, in this casearound −60 mV. To achieve this it is necessary to either co-express anion channel, such as a potassium inward rectifier, that can maintain theresting transmembrane potential of the cell to around −90, mV, oridentify a cell line that endogenously expresses similar ion channels.Cell types of this type include RBL cells and HEK-293 cells.

In other cases it may be necessary to use the expression of a second ionchannel, in conjunction with electrical stimulation to drive the cellmembrane to a specific transmembrane potential, to enable the first ionchannel of interest to be assayed. Examples of this situation occur whenassaying non-voltage regulated ion channels such as ligand-gatedchannels. Co-expression of a voltage regulated sodium channel, forexample in conjunction with electrical stimulation can be used to setthe transmembrane potential to transmembrane potentials of between about+10 to +60 mV. By comparison, co-expression of voltage regulatedpotassium channels in conjunction with electrical stimulation can setthe transmembrane potential to transmembrane potentials of between about−90 to −30 mV. These approaches thus enable the effective manipulationof the transmembrane potential over a relatively wide range therebyenabling the analysis of virtually any ion channel.

Typically when using this co-expression approach it is necessary tore-screen any hits obtained with the cell line co-expressing both ionchannels, with the cell line expressing only the ion channel used to setthe transmembrane potential. This enables drugs that affect this secondion channel to be differentiated from those that actually influence theion channel of interest. Alternatively selective toxins such as TTX canbe used to selectively inhibit one class of ion channel.

b. b) Transfection of Ion Channels

Nucleic acids used to transfect cells with sequences coding forexpression of the ion channel of interest are typically in the form ofan expression vector including expression control sequences operativelylinked to a nucleotide sequence coding for expression of the channel. Asused, the term “nucleotide sequence coding for expression of a channel”refers to a sequence that, upon transcription and translation of mRNA,produces the channel. This can include sequences containing, e.g.,introns. As used herein, the term “expression control sequences” refersto nucleic acid sequences that regulate the expression of a nucleic acidsequence to which it is operatively linked. Expression control sequencesare operatively linked to a nucleic acid sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleic acid sequence. Thus, expressioncontrol sequences can include appropriate promoters, enhancers,transcription terminators, a start codon (i.e., ATG) in front of aprotein-encoding gene, splicing signals for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of themRNA, and stop codons.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the ion channel coding sequence,operatively coupled to appropriate localization or targeting domains andappropriate transcriptional/translational control signals. For exampleby reference to the sequence accession numbers, or references in Tables1 to 3, one or ordinary skill in the art can identify the sequence ofthe ion channel of interest. These methods include in vitro recombinantDNA techniques, synthetic techniques and in vivo recombination/geneticrecombination. (See, for example, the techniques described in Maniatis,et al., Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y., 1989). Many commercially available expression vectorsare available from a variety of sources including Clontech (Palo Alto,Calif.), Stratagene (San Diego, Calif.) and Invitrogen (San Diego,Calif.) as well as and many other commercial sources.

A contemplated version of the method is to use inducible controllingnucleotide sequences to produce a sudden increase in the expression ofthe ion channel of interest e.g., by inducing expression of the channel.Example inducible systems include the tetracycline inducible systemfirst described by Bujard and colleagues (Gossen and Bujard (1992) Proc.Natl. Acad. Sci USA 89 5547-5551, Gossen et al. (1995) Science 2681766-1769) and described in U.S. Pat. No. 5,464,758.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells that arecapable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also beco-transfected with DNA sequences encoding the ion channel, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express the ionchannel. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982). Preferably, a eukaryotic host is utilized as thehost cell as described herein.

Selection of stable clones will typically be made on the basis ofsuccessful expression of the ion channel of interest at sufficient levelto enable it's facile detection. In many cases this analysis willrequire functional characterization of individual clones to identifythose that exhibit appropriate electrophysiological characteristicsconsistent with expression of the clone of interest. This analysis canbe completed via the use of patch clamping, or via the measurement oftransmembrane potentials using transmembrane potential sensitive dyes asdescribed below. An advantage to the use of this latter method is thatit is compatible with fluorescence activated cell sorting and providesfor the rapid analysis of many thousands of individual clones persecond. In some cases where the sodium channel is electrically silent inthe resting cell, confirmation of expression can also be readilyachieved by immunochemistry using antibodies raised against the nativeion channel, or a defined epitope introduced in the ion channel viamolecular techniques as described above.

In cases where cells are transfected with a first ion channel ofinterest, and a second ion channel to set the transmembrane potential,optimization of the relative expression of both ion channels isimportant. Typically the optimal relative expression of the two ionchannels is determined empirically by selecting clones that provide themaximum dynamic range and minimal statistical variation in theirresponse.

7. VII Measurement of Transmembrane Potentials

Transmembrane potential changes and the measurement of specific ionchannels conductance via the use of the present invention can bedetected by use of any of the known means of measuring transmembranepotential or ion movement. These methods include, for example, patchclamping (Hamill et al, Pfluegers Arch. 391:85-100, 1981), FRET basedvoltage sensors, electrochromic transmembrane potential dyes (Cohen etal., Annual Reviews of Neuroscience 1: 171-82, 1978), transmembranepotential redistribution dyes (Freedman and Laris, Spectroscopicmembrane probes Ch 16, 1988), extracellular electrodes (Thomas et al.,Exp. Cell Res. 74: 61-66, 1972), field effect transistors (Fromherz etal., Science 252: 1290-1293, 1991), radioactive flux assays, ionsensitive fluorescent or luminescent dyes, ion sensitive fluorescent orluminescent proteins, the expression of endogenous proteins or the useof reporter genes or molecules.

Preferred methods of analysis for high throughput screening typicallyinvolve the use of optical readouts of transmembrane potential, or ionchannel conductance. Such methods include the use of transmembranepotential or ion sensitive dyes, or molecules, that typically exhibit achange in their fluorescent or luminescent characteristics as a resultof changes in ion channel conductance or transmembrane potential.

A preferred optical method of analysis for use with the presentinvention has been described in U.S. Pat. No. 5,661,035 issued Aug. 26,1997, hereby incorporated by reference). This approach typicallycomprises two reagents that undergo energy transfer to provide aratiometric fluorescent readout that is dependent upon the transmembranepotential. Typically the approach uses a voltage sensing lipophilic dyeand a voltage insensitive fluorophore associated with a cell membrane.(see González et al. Drug Discovery Today 4:431-439, 1999).

In one embodiment, two dye molecules, a coumarin-linked phospholipid(CC2-DMPE) and an oxonol dye such as bis-(1,2-dibutylbarbituric acid)trimethine oxonol [DiSBAC₄(3)], are loaded into the plasma membrane ofcells. CC2-DMPE partitions into the outer leaflet of the plasma membranewhere it acts as a fixed FRET donor to the mobile, voltage sensitiveoxonol acceptor. Cells with relatively negative potentials inside willpush the negatively charged oxonol to the outer leaflet of the plasmamembrane, resulting in efficient FRET (i.e. quenching of the coumarindonor and excitation of the oxonol acceptor). Depolarization results inrapid translocation of the oxonol to the inner surface of the plasmamembrane, decreasing FRET. Because FRET can only occur over distances ofless than 100 Å, excitation of the coumarin results in specificmonitoring of oxonol movements within the plasma membrane.

The response times for these assays is readily altered by increasing ordecreasing the hydrophobicity of the oxonol. For example, the morehydrophobic dibutyl oxonol DiSBAC₄(3) has a time constant ofapproximately 10 ms, significantly faster than the less hydrophobicdiethyl oxonol DiSBAC₂(3).

Loading of the dyes is typically achieved at room temperature prior tothe start of transmembrane potential measurements. Typically cells areloaded sequentially with the coumarin lipid followed by the oxonol.Typical loading concentrations for coumarin lipids range from about 4 to15 μM (final concentration) and staining solutions are typicallyprepared in Hanks Balanced salt solution with 10 mM HEPES, 2 g/L glucoseand about 0.02% Pluronic-127 at a pH of around 7.2 to 7.4. Loading isusually acceptable after about 30 minutes incubation, after which excessdye may be removed if desired. Oxonol dyes are typically loaded at aconcentration between 2 and 10 μM for 25 minutes at room temperature,the more hydrophobic DiSBAC₄(3) is usually loaded in the presence of 2-3μM Pluronic-127. Optimal loading concentrations vary between cell typesand can be empirically determined by routine experimentation. Typicallysuch optimization experiments are conducted by systematically titratingthe concentrations of the first reagent, and then for each concentrationtested, titrating the concentration of the second reagent. In this wayit is possible to obtain both the optimal loading concentrations foreach reagent, and the optimal relative ratio to achieve a maximal signalto noise ratio.

In some cases it may be preferred to add, or load one, or more of theFRET reagents with one or more light absorbing substances in order toreduce undesired light emission, as for example described in commonlyowned U.S. patent application Ser. No. 09/118,497, filed Jul. 17, 1998;U.S. patent application Ser. No. 09/120,516, filed Jul. 21, 1998, andU.S. patent application Ser. No. 09/122,477 filed Jul. 23, 1998.

FRET based voltage sensors may also be derived from the use of othermembrane targeted fluorophores in conjunction with a mobile hydrophobicdonor or acceptor. Other such compositions are disclosed, for example,in U.S. patent application Ser. No. 09/459,956, filed Dec. 13, 1999.

Suitable instrumentation for measuring transmembrane potential changesvia optical methods includes microscopes, multiwell plate readers andother instrumentation that is capable of rapid, sensitive ratiometricfluorescence detection. A preferred instrument of this type is describedin U.S. patent application Ser. No. 09/118,728 filed Jul. 17, 1998. Thisinstrument (the Voltage/Ion Probe Reader or VIPR™) is an integratedliquid handler and kinetic fluorescence reader for 96-well and greatermultiwell plates. The VIPR™ reader integrates an eight channel liquidhandler, a multiwell positioning stage and a fiber-optic illuminationand detection system. The system is designed to measure fluorescencefrom a column of eight wells simultaneously before, during and after theintroduction of liquid sample obtained from another microtiter plate ortrough. The VIPR™ reader excites and detects emission signals from thebottom of a multiwell plate by employing eight trifurcated opticalbundles (one bundle for each well). One leg of the trifurcated fiber isused as an excitation source, the other two legs of the trifurcatedfiber being used to detect fluorescence emission. A ball lens on the endof the fiber increases the efficiency of light excitation andcollection. The bifurcated emission fibers allow the reader to detecttwo emission signals simultaneously and are compatible with rapidsignals generated by the FRET-based voltage dyes. Photomultiplier tubesthen detect emission fluorescence, enabling sub-second emission ratiodetection.

8. VIII Stimulation Protocols

In one aspect, the present invention includes methods for modulating thetransmembrane potentials of living cells via electrical stimulation, andthe use of these methods for assaying the activity of virtually any ionchannel or transporter system.

a. a) Measurement of Specific Channel Conductances

(a) 1. Assay of Sodium Channels

A variety of different isoforms of mammalian voltage dependent sodiumchannels have been identified, and are summarized below in Table 1.These channels can be classified into three main groups (for review seeGoldin, Annals N.Y. Academy of Sciences 868:38-50, 1999).

TABLE 1 Sodium Channel Sub-type Summary Channel Name & Sub-type/ TissueAccession Gene Symbol Alternate names Distribution Number SCN1A (Nav1.1)Rat I (rat) CNS/PNS X03638 HBSCI (human) CNS X65362 GPB1 (Guinea pig)CNS AF003372 SCN2A (Nav1.2) Rat II (rat) CNS X03639 HBSCII (human) CNSX65361 HBA (human) CNS M94055 Nav 1.2A Rat IIA CNS X61149 SCN3A (Nav1.3) Rat III (rat) CNS Y00766 SCN4A (Nav1.4) SkM1, μl (rat) skeletalmuscle M26643 SkM1 (human) Skeletal muscle M81758 SCN5A (Nav1.5) SkM2(rat) skeletal M27902 RH1(rat) muscle/heart H1(human) heart M77235 SCN8A(Nav1.6) NaCh6 (rat) CNS/PNS L39018 PN4a (rat) CNS/PNS AF049239A Scn8a(mouse) CNS U26707 Scn8a (human) CNS AF050736 CerIII (Guinea pig) CNSAF003373 SCN9A (Nav1.7) PN1 (rat) PNS U79568 HNE-Na (human) thyroidX82835 Nas (rabbit) Schwann cells U35238 SCN10A Nav1.8 SNS (rat) PNSX92184 PN3 (rat) PNS U53833 SNS (mouse) PNS Y09108 SCN6A Nav2.1 Na2.1(human) Heart, uterus M91556 muscle SCN7A Nav2.2 Na-G (rat) astrocytesM96578 SCL11(rat) PNS Y09164 Nav2.3 Na2.3 (mouse) Heart, uterus L36179muscle Nav3.1 NaN (rat) PNS AF059030 SCN1B Naβ1.1 β-1 (rat) CNS M91808β-1 (human) CNS L10338 SCN2B Naβ2.1 β-2 (rat) CNS U37026 β-2 (human) CNSAF007783

The voltage-dependent sodium channels in Table 1 vary widely in theirvoltage dependency and inactivation and activation kinetics.Voltage-gated sodium channels have many different conformations, whichcan be classified into three states. (1) The resting state, in which thechannel is closed and no current can flow. This is the typical statewhen a sodium channel is expressed in a cell with a restingtransmembrane potential of below about −60 mV. The channel can berapidly driven into the open state by depolarization, usually to atransmembrane potential of above about −50 mV. (2) The activated state,in which the channel is open and ions can pass through. Because theintracellular concentration of sodium is low in a normal resting cell,while the extracellular concentration is high, sodium ions flow into thecell and drive the transmembrane potential more positive. The open statehas a short lifetime, generally on the order of one millisecond, afterwhich it passes into the inactivated state. (3) The inactivated state,in which a channel has closed and ions can not pass through the channel.The channel cannot be directly opened once in the inactivated state. Itwill first go to the resting state, which occurs if the transmembranepotential is held very negative (generally below −80 mV) for severalmilliseconds. The time constants and threshold potentials fortransitions between these three states vary greatly between channelsubtypes.

During these experiments, the response will be compared for cells withactive channels, and for cells in which the channels arepharmacologically blocked. If a suitable pharmacological agent is notavailable, the blocked state can be emulated with an un-transfected cellline. The optimal stimulus parameters will yield the smallestcoefficient of variation of the difference in signals of the two cellpopulations.

(i) i) Assays for Voltage-Dependent Sodium Channels in an InactivatedState

Preferred cells include those with resting transmembrane potentialsabove the activation threshold for the ion channel of interest, and inwhich there are no other ion channels expressed. Cells meeting thesecriteria include CHL and LTK(−) cells. After choosing a target ionchannel, cells are transfected and clones are selected as described insection III. Alternatively, a cell line that endogenously expresses thechannel of interest, and low levels of other channels, could be used.For example, the CHO-K1 cell line expresses a voltage-gated sodiumchannel, and very low levels of other ion channels. Cells are platedinto multiwell microtiter plates, cultured, and stained withvoltage-sensitive dyes as described in section IV prior to initiatingelectrical stimulation. Initial experiments are typically carried out ina 96-well multiwell plate, with an equal number of cells in each well.Generally columns of eight wells are simultaneously stimulated underidentical conditions to provide statistically significant data on thevariation in cellular response.

An optimal electrical stimulation protocol should hyperpolarize part ofthe plasma membrane of the majority of the cells long enough to releasethe sodium channels from inactivation, prior to providing an activatingdepolarization, without electroporating or killing the cells. Typicallythis requires sustained transmembrane potentials of around −60 to −80 mVfor periods ranging from about 0.5 to about 20 ms to be created withinthe cell.

A preferred stimulation protocol that achieves this effect is biphasic,so that ion channels present on both the extreme edges of the cells arereleased from inactivation as the biphasic waveform reverses polarity.Typically one would start out with initial conditions using a biphasicsquare wave kernel of 5 msec per phase and an amplitude of 25 V/cm. Thekernel would be repeated at a regular rate of about 20 Hz for a totaltrain duration of about three seconds. One would then optimize the pulseamplitude (up to a maximum of about 60 V/cm), duration (in the range of0.1 to 50 ms), and then frequency (in the range of 0 to 1 kHz). Ifnecessary changes in the pulse shape could also be explored to determineif these resulted in more efficient electrical stimulation. The optimalstimulus parameters will yield the maximum cellular stimulation(compared to cells with the channel blocked, or not present) withsmallest coefficient of variation of the signal among the different testwells, at the lowest electric field strength, and at the lowest dutycycle for passage of current through the electrodes. After a particularset of parameters is chosen, a titration of staining concentrations forthe voltage sensor dye(s) should be performed as described above, tofurther optimize the signal size and coefficient of variation of theresponses. These procedures (dye concentrations, electric fieldstrength, and stimulus duration and frequency) can be iterated tofurther optimize the signal.

(ii)

(iii)ii) Assays for Sodium Channels Normally in the Resting State

Preferred cells include those with resting transmembrane potentialsbelow the activation threshold for the ion channel of interest, and inwhich the expression of other ion channels is largely confined to a fewcharacterized ion channel types. Cells of this type include HEK-293 andRBL cells as well as F11 and HL5 cells. After choosing a target ionchannel, cells are transfected with the ion channel of interest andclones are selected as described above. Alternatively, as in the case ofF 11 and HL5 cells, endogenous sodium channels can be used. Afterselection and characterization, cell clones are plated into multiwellmicrotiter plates and stained with voltage-sensitive dyes as describedabove. As previously, initial experiments are typically carried out in a96-well multiwell plate, with an equal number of cells in each well.Generally columns of eight wells are simultaneously stimulated underidentical conditions to provide statistically significant data on thevariation in cellular response.

A number of assay approaches are possible depending on the expressionlevel of the sodium channel of interest in the cell. For high levels ofvoltage-dependent sodium channel expression, the sodium current can belarge enough to create a large transmembrane potential change after asingle channel activation/inactivation sequence. In these cases smallpositive perturbations in the transmembrane potential created viaelectrical stimulation can be sufficient to activate enough sodiumchannels that the subsequent sodium ion entry depolarizes the entirecell thereby activating all the sodium channels. The stimulus fieldshould typically be applied for a time long enough to activate thechannels, but not so long as to interfere with the subsequent ion flux.After the cell transmembrane potential has re-polarized, the stimulationprocedure can be repeated. Subsequent stimulation events can beidentical to the first, or varied to examine time-dependent propertiesof the channels.

Typically one would start out with initial conditions using a biphasicsquare wave kernel of 500 μs per phase and an amplitude of 10 V/cm. Onewould then optimize the pulse amplitude (between 5 and 60 V/cm) andduration (between 0.1 and 1 ms). If necessary changes in the pulse shapecould also be explored to determine if these resulted in more efficientelectrical stimulation. The optimal stimulus parameters will yield themaximum cellular stimulation with smallest coefficient of variation ofthe signal among the different test wells, at the lowest electric fieldstrength, and at the lowest duty cycle for passage of current throughthe electrodes. After a particular set of parameters is chosen, atitration of staining concentrations for the voltage sensor dye(s)should be performed as described above, to further optimize the signalsize and coefficient of variation of the responses. These procedures(dye concentrations, electric field strength, and stimulus duration andfrequency) can be iterated to further optimize the signal.

Often it will be necessary to use cells whose expression of sodiumchannels is too low to give acceptable signal sizes from single stimuli.It may also be desirable to maintain a large signal over an extendedperiod of time. In these cases, the cells can be given pulse trains asdescribed for channels held above the activation potential. Withbiphasic stimulus pulses, the sodium channels can be activatedindependent of the starting transmembrane potential. By keeping theinter-pulse interval shorter than the membrane time constant, eachstimulus will drive current into the cell until an equilibrium betweeninward and outward currents is established. This voltage deviation willbe maintained as long as the stimulus train continues.

The stimulation protocols in this case are essentially the same asdescribed for cells whose resting potential is above the inactivationthreshold. In general, a series of initial experiments are conductedusing a biphasic square wave kernel repeated at a regular rate for afixed train duration. The pulse duration varies from about 1 μs to about1 s, and more preferably from about 100 μs to about 20 ms. The pulseamplitude varies from 0 V/cm to about 60 V/cm, and more preferably from10 V/cm to 50 V/cm. The frequency of stimulation varies between 0 Hz(i.e. a single pulse) and 100 kHz, and more preferably from 0 Hz toabout 1 kHz. The pulse train varies between 0 s (i.e. a single pulse)and about 100 s, and more preferably between 0 s and 10 s. The optimalstimulus parameters will yield the maximum transmembrane potentialchanges (compared to cells with the channel blocked, or not present) andsmallest coefficient of variation of the signal among the test wells, atthe lowest electric field strength. After a particular set of parametersis chosen, a titration of staining concentrations for the voltage sensordye(s) is typically performed as described above to further optimize thesignal size and coefficient of variation of the responses. Theseprocedures (dye concentrations, electric field strength, and stimulusduration and frequency) can be iterated to further optimize the signal.

(b)

(c) b) Potassium Channels

Voltage-dependent potassium channels repolarize nerve and muscle cellsafter action potential depolarization. They also play importantregulatory roles in neural, muscular, secretory, and excretory systems.Most cells actively maintain a high intracellular potassiumconcentration, so that the reversal transmembrane potential forpotassium is around −90 mV. Potassium typically flows out of the cell,so that opening more potassium-selective channels tends to drive thetransmembrane potential more negative, in contrast to sodium channelopening that typically drives the transmembrane potential more positive.

A summary of the numerous potassium sub-types is presented in Table 2below.

TABLE 2 Potassium Channel Sub-type Summary Accession Channel TypeSub-type/Alternate names Number Reference ATP regulated rKir1.1 (ROMK1)(rat) U12541 U.S. Pat. No. 5,356,775 hKir1.1 (ROMK1) U.S. Pat. No.(human) 5,882,873 Kir1.2 U73191 Kir1.3 U73193 II. β-cell U.S. Pat. No.5,744,594 III. hβIR U.S. Pat. No. 5,917,027 IV. HuK_(ATP)-1 EP 0 768 379A1 Constitutively Active Kir2.1(IRK1) U12507 U.S. Pat. No. 5,492,825U.S. Pat. No. 5,670,335 Kir2.2 X78461 Kir2.3 U07364 G-protein RegulatedKir3.1 (GIK1, KGA) U01071 U.S. Pat. No. 5,728,535 Kir3.2 U11859 U.S.Pat. No. 5,734,021 Kir3.3 U11869 U.S. Pat. No. 5,744,324 Kir3.4 (CIR)X83584 U.S. Pat. No. 5,747,278 Kir4.1(BIR10) X83585 Kir5.1(BIR9) X83581Kir6.1 D42145 Kir6.2 D5081 Kir7.1 EP 0 922 763 A1 Voltage RegulatedKCNA1 hKv1.1 (RCK1, RBK1, LO2750 MBK1, MK1, HuK1) KCNA2 Kv1.2 (RBK2,RBK5, NGK1, HuKIV) KCNA3 Kv1.3 (KV3, RGK5, HuKIII, HPCN3, KCNA4 Kv1.4(RCK4, RHK1, HuKII) KCNA5 Kv1.5 (KV1, HPCN1, HK2) KCNA6 Kv1.6 (KV2,RCK2, HBK2) KCNA7 Kv 1.7 (MK6, RK6, U.S. Pat. No. HaK6) 5,559,009 Kν2(Shab) KCNB1 Kv2.1 (DRK1, mShab) M64228 KCNB2 Kv2.2 (CDRK1) K channel 2U.S. Pat. No. 5,710,019 Kν3 (Shaw) KCNC1 Kv3.1 (NGK2) KCNC2 Kv3.2(RKShIIIA) KCNC3 Kv3.3 (KShIIID) X60796 KCNC4 Kv3.4 (Raw3) Kν4 (Shal)KCND1 Kv4.1 (mShal, KShIVA) M64226 KCND2 Kv4.2 (RK5, Rat Shal 1) KCND3Kv4.3 (KShIVB) hKv5.1 (IK8) WO 99/41372 Kv6.1 (K13) Kv7 Kv8.1 Kv9Delayed Rectifier KvLQT1 AF000571 U.S. Pat. No. 5,599,673 HERG (erg)U04270 PCT WO99/20760 Calcium regulated Ca²⁺ Regulated Big BKCa (hSLO)U11717 HBKb3 (β-subunit) PCT WO99/42575 Maxi-K U.S. Pat. No. 5,776,734U.S. Pat. No. 5,637,470 Ca²⁺ Regulated-small KCNN1 SKCa1 U69883 KCNN2SKCa2 U69882 KCNN3 SKCa3 U69884 KCNN4 SKCa4 (IKCa1) Muscle Nerve 199922(6) 742-50 TWIK1 U33632

Potassium channels show enormous diversity in terms of activation andinactivation time constants and voltage dependencies. In general,voltage-dependent potassium channels show voltage dependence similar tosodium channels, being closed at very negative potentials and openingabove a certain threshold. Potassium channels may have multiple restingstates, multiple inactivated states, and typically a single activatedstate. Unlike voltage-dependent sodium channels, transitions are allowedbetween most states. These transitions are activation (moving from aresting to the open state), deactivation (moving from the open state toa resting state), inactivation (moving from a resting or open state toan inactivated state), release from inactivation (moving from aninactivated state to a resting state), and flickering (moving from aninactivated state to the open state). There is a great diversity in thethresholds of the transitions, and in the voltage dependencies of thetransition rates. Activation time constants range from 0.1 to 1000 mswith threshold activation potentials from −80 to +20 mV. Inactivationtime constants range from 0.1 to infinity (i.e. no inactivation) withthreshold potentials from −60 to 0 mV. Time constants for release frominactivation range from 0.5 ms to 100 ms with threshold potentials from−70 to 0 mV.

Stimulus protocols necessary to obtain measurable channel-dependentsignals are somewhat dependent upon the specific properties of thechannel in question. Because of the diversity in parameters involtage-dependent potassium channels, the optimization of an electricalstimulation protocol may take several iterations.

During these experiments, the response will be compared for cells withactive channels, and for cells in which the channels arepharmacologically blocked. If a suitable pharmacological agent is notavailable, the blocked state can be emulated with an un-transfected cellline. The optimal stimulus parameters will yield the smallestcoefficient of variation of the difference in signals of the two cellpopulations.

i) Assays Using Direct Stimulation of the Potassium Channel

1) Voltage Regulated Potassium Channels

Because potassium channels generate outward currents, activating thechannels causes negative transmembrane potential changes. Underphysiological conditions, the reversal potential for potassium is around−90 mV. Because cells expressing only a voltage-dependent potassiumchannel generally have resting potentials near the activation threshold,direct stimulation should work for those voltage-dependent potassiumchannels which have activation thresholds above about −50 mV. Whilesmall negative deflections in the transmembrane potential (less than 40mV change) can be reliably detected using the FRET voltage-sensitivedyes, it is often preferable to perform high-throughput screens withlarger signals.

Preferred cell types include those cells that express a minimal level ofother ion channels, such as CHO-K1, CHL, and LTK(−). The transfectionand selection of clones expressing ion channels of interest willgenerally be performed as described above for sodium ion channelsnormally in the resting state. Alternatively, a cell line whichendogenously expresses the channel of interest could be used. Thelabeling and measurement of cells with transmembrane potential dyes willgenerally be performed as described for sodium ion channels normally inthe resting state.

The stimulation protocol will advantageously depolarize part of theplasma membrane long enough to activate the voltage-dependent potassiumchannels. Unlike the case for voltage-dependent sodium channels,voltage-dependent potassium channels will typically pass current duringthe depolarizing phase of the stimulus pulse. On the side of the cellwhere the transmembrane potential is driven in a negative direction, thepotassium channels release from inactivation (if the channel in questionexperiences voltage-dependent inactivation). On the side of the cellwhere the transmembrane potential is driven in a positive direction,potassium channels activate and pass outward current. Thus, the stimuluspulse duration should not greatly exceed the inactivation time. Thepotassium current tends to drive the average transmembrane potentialnegative of the resting potential. After the stimulus pulse, thetransmembrane potential will exponentially relax to the restingpotential. By repeating the stimulus after a time shorter than themembrane time constant, the average cell membrane can be driven furthernegative. Using a train of stimuli, a large and sustained signal can beobtained.

A preferred stimulation protocol that achieves this effect is biphasic,so that ion channels present on both the extreme edges of the cells canparticipate in enabling potassium ion movement. Typically one wouldstart out with initial conditions using a biphasic square wave kernel of5 msec per phase and an amplitude of 25 V/cm. The kernel would berepeated at a regular rate of about 20 Hz for a total train duration ofabout three seconds. One would then optimize the pulse amplitude,duration, and then frequency. If necessary changes in the pulse shapecould also be explored to determine if these resulted in more efficientelectrical stimulation. The optimal stimulus parameters will yield themaximum average transmembrane potential change (compared to cells withthe channel blocked, or not present) with smallest coefficient ofvariation of the signal among the different test wells, at the lowestelectric field strength, and at the lowest duty cycle for passage ofcurrent through the electrodes. After a particular set of parameters ischosen, a titration of staining concentrations for the voltage sensordye(s) should be performed as described above, to further optimize thesignal size and coefficient of variation of the responses. Theseprocedures (dye concentrations, electric field strength, and stimulusduration and frequency) can be iterated to further optimize the signal.

(a) 2) Inward-Rectifier Potassium Channels

Contrary to its name, the function of the inward rectifier channel isnot to allow potassium into the cell. Inward flow of potassium can onlyoccur (1) when the transmembrane potential falls below the potassiumequilibrium potentials, or (2) if the extracellular potassiumconcentration rises. Neither situation normally occurs, because (1)under normal physiological conditions, since potassium is the ion withthe most negative reversal potential, no ionic current can drive thepotential more negative than the potassium reversal potential, and (2)except under pathological conditions, the extracellular potassiumconcentration is tightly controlled. However, using electricalstimulation, parts of the cell membrane can be driven below VK,promoting potassium ion entry into the cell. This will cause a netpositive transmembrane potential change and can be detected as apositive signal. To develop and optimize an assay for blockers of theinward rectifier, one could therefore follow the following procedure.

Preferred cell types include those cells that express a minimal level ofother ion channels, such as CHO-K1, CHL, and LTK(−). The transfectionand selection of clones expressing ion channels of interest willgenerally be performed as described above for sodium ion channelsnormally in the resting state. Alternatively, a cell line whichendogenously expresses the channel of interest could be used. Thelabeling and measurement of cells with transmembrane potential dyes willgenerally be performed as described for sodium ion channels normally inthe resting state.

A preferred stimulation protocol uses a biphasic kernel, so that ionchannels present on both the extreme edges of the cells participate.Typically one would start out with initial conditions using a biphasicsquare wave kernel of 5 msec per phase and an amplitude of 25 V/cm. Thekernel would be repeated at a regular rate of about 20 Hz for a totaltrain duration of about three seconds. One would then optimize the pulseamplitude, duration, and then frequency. If necessary changes in thepulse shape could also be explored to determine if these resulted inmore efficient electrical stimulation. The optimal stimulus parameterswill yield the maximum cellular stimulation (compared to cells with thechannel blocked, or not present) with the smallest coefficient ofvariation of the signal among the different test wells, at the lowestelectric field strength, and at the lowest duty cycle for passage ofcurrent through the electrodes. After a particular set of parameters ischosen, a titration of staining concentrations for the voltage sensordye(s) should be performed as described above, to further optimize thesignal size and coefficient of variation of the responses. Theseprocedures (dye concentrations, electric field strength, and stimulusduration and frequency) can be iterated to further optimize the signal.

(i) iii) Assays Using a Voltage-Dependent Sodium Counter-Channel

This method involves the use of a cell line expressing thevoltage-dependent potassium channel of interest and which also expressesa voltage-dependent sodium channel. In this method the approach is touse electrical stimulation protocols designed to specifically activatethe voltage dependent sodium channel. In this case electricalstimulation causes sodium ions to enter the cell, causing a positivevoltage change. The presence of the potassium channel of interest willtend to suppress the positive response of the sodium channel by allowingpotassium ions to leave the cell. The assay takes advantage of theabsence of outward current when a test chemical blocks the potassiumchannel, thereby restoring the large positive voltage response normallyinduced by activation of the sodium channels. The optimization of thebalance of currents is important in this method to ensure that the assayis sensitive to potassium channel blockade. If the sodium current is toosmall relative to the potassium current, the dose-response curve for thepotassium channel blocker will be shifted towards higher concentrations.For example, in the extreme case where the potassium current is 100times larger than the sodium current, 99% of the potassium channelswould have to be blocked in order to get a 50% response from the sodiumchannels.

Because this method involves driving a voltage-dependent sodium channelwith repetitive pulses, the protocol development is essentially the sameas described above for voltage-activated sodium channels in aninactivated state. Typically one would start out with initial conditionsusing a biphasic square wave kernel of 5 msec per phase and an amplitudeof 25 V/cm. The kernel would be repeated at a regular rate of about 20Hz for a total train duration of about three seconds. One would thenoptimize the pulse amplitude, duration, and then frequency. If necessarychanges in the pulse shape could also be explored to determine if theseresulted in more efficient electrical stimulation. The optimal stimulusparameters will yield the maximum cellular stimulation (compared tocells with the channel blocked, or not present) with smallestcoefficient of variation of the signal among the different test wells,at the lowest electric field strength, and at the lowest duty cycle forpassage of current through the electrodes. After a particular set ofparameters is chosen, a titration of staining concentrations for thevoltage sensor dye(s) should be performed as described above, to furtheroptimize the signal size and coefficient of variation of the responses.These procedures (dye concentrations, electric field strength, andstimulus duration and frequency) can be iterated to further optimize thesignal.

In this assay format, there will ideally be no (or a very small)response to stimulation in the absence of channel block, because thepotassium current will counteract the sodium current. Therefore, tooptimize the stimulus conditions, it will be necessary to compareresponses with and without the activity of the potassium channel.Ideally, this will be accomplished using a selective blocker of thepotassium channel. In those cases where such a blocker is yet unknown,it will be possible to use the cell line containing only the sodiumcounter-channel.

Because this assay format involves two ion channels, modulators ofeither channel will affect the voltage response. In this case, a hit (ablocker of the potassium channel) will restore the voltage response. Thescreening format automatically ignores compounds which block only thesodium channel. However, stimulation of the cells in the presence ofcompounds which block both channels will also result in no voltagedeflection, suggesting that the compound is inactive. Because compoundsof this type may be of interest, a method to unmask them is alsoavailable. By performing the identical compound screen using the parentcell line, which contains the sodium channel but not the potassiumchannel, blockers of the sodium channel can be found. Compounds whichare found to block the sodium channel can then be tested separately tofind if they have activity against the potassium channel.

(b) c) Assay of Calcium Channels

Calcium channels are generally found in many cells where, among otherfunctions, they play important roles in signal transduction. Inexcitable cells, intracellular calcium supplies a maintained inwardcurrent for long depolarizing responses and serves as the link betweendepolarization and other intracellular signal transduction mechanisms.Like voltage-gated sodium channels, voltage-gated calcium channels havemultiple resting, activated, and inactivated states.

Multiple types of calcium channels have been identified in mammaliancells from various tissues, including skeletal muscle, cardiac muscle,lung, smooth muscle and brain, [see, e.g., Bean, B. P. (1989) Ann. Rev.Physiol. 51:367-384 and Hess, P. (1990) Ann. Rev. Neurosci. 56:337]. Thedifferent types of calcium channels have been broadly categorized intofour classes, L-, T-, N-, and P-type, distinguished by current kinetics,holding potential sensitivity and sensitivity to calcium channelagonists and antagonists. Four subtypes of neuronal voltage-dependentcalcium channels have been proposed (Swandulla, D. et al., Trends inNeuroscience 14:46, 1991).

The cDNA and corresponding amino acid sequences of the α1, α2, β and γsubunits of the rabbit skeletal muscle calcium channel have beendetermined [see, Tanabe et al. (1987) Nature 328:313-318; Ruth et al.(1989) Science 245:1115-1118; and U.S. Pat. No. 5,386,025]. In addition,the cDNA and corresponding amino acid sequences of α1 subunits of rabbitcardiac muscle [Mikami, A. et al. (1989) Nature 340:230-233] and lung[Biel, M. (1990) FEBS Letters 269:409-412] calcium channels have beendetermined. In addition, cDNA clones encoding a rabbit brain calciumchannel (designated the BI channel) have been isolated [Mori, Y. et al.(1991) Nature 350:398-402].

Partial cDNA clones encoding portions of several different subtypes,referred to as rat brain class A, B, C and D, of the calcium channel α1subunit have been isolated from rat brain cDNA libraries [Snutch, T. etal. (1990) Proc. Natl. Acad. Sci. USA 87:3391-3395]. More recentlyfull-length rat brain class A [Starr, T. et al. (1991) Proc. Natl. Acad.Sci. USA 88:5621-5625] and class C [Snutch, T. et al. (1991) Neuron7:45-57] cDNA clones have been isolated. Although the amino acidsequence encoded by the rat brain class C DNA is approximately 95%identical to that encoded by the rabbit cardiac muscle calcium channelα1 subunit-encoding DNA, the amino acid sequence encoded by the ratbrain class A DNA shares only 33% sequence identity with the amino acidsequence encoded by the rabbit skeletal or cardiac muscle α1subunit-encoding DNA. A cDNA clone encoding another rat brain calciumchannel α1 subunit has also been obtained [Hui, A. et al. (1991) Neuron7:35-44]. The amino acid sequence encoded by this clone is approximately70% homologous to the proteins encoded by the rabbit skeletal andcardiac muscle calcium channel DNA. A cDNA clone closely related to therat brain class C α1 subunit-encoding cDNA and sequences of partial cDNAclones closely related to other partial cDNA clones encoding apparentlydifferent calcium channel α1 subunits have also been isolated [seeSnutch, T. et al. (1991) Neuron 7:45-57; Perez-Reyes, E. et al. (1990)J. Biol. Chem. 265:20430; and Hui, A. et al. (1991) Neuron 7:35-44].

For known calcium channels that have been characterized, activation timeconstants range from 0.1 to 10 ms with threshold potentials from −80 to−20 mV. Inactivation time constants range from 0.1 to ∞ (i.e. noinactivation) with threshold potentials from −60 to −20 mV. Timeconstants for release from inactivation range from 0.5 ms to 100 ms withthreshold potentials from −70 to −40 mV.

Choice of cell line and induction of voltage-dependent calcium currentsare performed using the general guidelines and approaches discussedabove for sodium channels. Preferred cell types include those cells thatexpress a minimal level of other ion channels, such as CHO-K1, CHL, andLTK(−). The transfection and selection of clones expressing ion channelsof interest will generally be performed as described above for sodiumion channels normally in the resting state. Alternatively, a cell linewhich endogenously expresses the channel of interest could be used. Thelabeling and measurement of cells with transmembrane potential dyes willgenerally be performed as described for sodium ion channels normally inthe resting state. Alternatively, the cells can be loaded withcalcium-sensitive fluorescent dyes such as Calcium Green, fluo3-AM, orindo-1.

In cells with low background currents, strong inward calcium currentscan be generated by driving portions of the membrane negative enough torelease the channels from inactivation. Then by reversing or releasingthe external electric field, the channels are exposed to potentialswhich activate the channels and permit calcium current to flow into thecell. The reversal potential for calcium in most cells is generally +60to +100 mV, so large voltage changes due to calcium influx are possible.We can use either membrane-bound voltage-sensitive dyes or intracellularcalcium dyes to monitor the activity of the cells. Due to the similarityin properties of calcium and sodium channels, the same general assayoptimization procedures outlined above for sodium channels will apply tocalcium channels.

Typically one would start out with initial conditions using a biphasicsquare wave kernel of 5 msec per phase and an amplitude of 25 V/cm. Thekernel would be repeated at a regular rate of about 20 Hz for a totaltrain duration of about three seconds. One would then optimize the pulseamplitude, duration, and then frequency. If necessary changes in thepulse shape could also be explored to determine if these resulted inmore efficient electrical stimulation. The optimal stimulus parameterswill yield the maximum cellular stimulation (compared to cells with thechannel blocked, or not present) with the smallest coefficient ofvariation of the signal among the different test wells, at the lowestelectric field strength, and at the lowest duty cycle for passage ofcurrent through the electrodes. After a particular set of parameters ischosen, a titration of staining concentrations for the voltage sensordye(s) should be performed as described above, to further optimize thesignal size and coefficient of variation of the responses. Theseprocedures (dye concentrations, electric field strength, and stimulusduration and frequency) can be iterated to further optimize the signal.

During these experiments, the response will be compared for cells withactive channels, and for cells in which the channels arepharmacologically blocked. If a suitable pharmacological agent is notavailable, the blocked state can be emulated with an un-transfected cellline. The optimal stimulus parameters will yield the smallestcoefficient of variation of the difference in signals of the two cellpopulations.

(c) d) Assay of Voltage-Dependent Chloride Channels

Chloride channels are found in the plasma membranes of virtually everycell in the body. Chloride channels mediate a variety of cellularfunctions including regulation of transmembrane potentials andabsorption and secretion of ions across epithelial membranes. Whenpresent in intracellular membranes of the Golgi apparatus and endocyticvesicles, chloride channels also regulate organelle pH. For a review,see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

Three distinct classes of chloride channels are apparent based on theirtype of regulation and structural conformation, Table 3. The first classincludes the GABA and Glycine receptor super families, the second classincludes the CFTR (Cystic fibrosis Transmembrane Conductance Regulator)and the third class includes the voltage regulated chloride channels.

TABLE 3 ii. Chloride Channel Sub-type Summary Tissue Channel TypeSub-type Distribution Reference Ligand gated GABA_(A) Receptor CNS & PNSSynapse 21, 189-274 family (1995) Glycine Receptor CNS & PNS TrendsNeurosci. 14, family 458-461 (1991) cAMP regulated CFTR EpithelialScience 245, tissues 1066-1073 (1989) Voltage regulated ClC-1 SkeletalNature 354, 301-304 Muscle (1991) ClC-2 Ubiquitous Nature 356, 57-60(1992) ClC-Ka Kidney J. Biol. Chem. 268, 3821-3824 (1993) ClC-Kb KidneyP.N.A.S. 91, 6943-6947 (1994) ClC-3 Broad, e.g. Neuron 12, 597-604Kidney & (1994) Brain ClC-4 Broad, e.g. Hum. Mol. Genet. 3 Kidney &547-552 (1994) Brain ClC-5 Mainly Kidney J. Biol. Chem 270, 31172-31177(1995) ClC-6 Ubiquitous FEBS. Lett. 377, 15-20 (1995) ClC-7 UbiquitousFEBS. Lett. 377, 15-20 (1995)

In contrast to ions like sodium and especially calcium, theelectrochemical gradient of chloride across the plasma membrane isgenerally not far from equilibrium. Thus, at the resting potential ofcells, the opening of chloride channels will not lead to largeexcursions of the plasma membrane voltage or dramatic changes inintracellular chloride concentrations. Because electrical stimulationtypically generates symmetrical voltage changes across the cellmembrane, no net chloride flux can be generated unless the conductivityof the channel is non-linear. For a linear leak conductance, a uniformelectric field will drive chloride into the cell on one side and out ofthe cell on the other side.

Direct electrical stimulation of chloride channels which have non-linearconductance curves (rectifiers) or voltage-activated gating can generatenet ion fluxes, which in turn will cause detectable transmembranepotential changes. Depending upon the voltage dependence of theconductance and gating, the transmembrane potential change can be eitherpositive or negative. For typical chloride channels (that activate atelevated potentials and close at more negative potentials) and foroutward rectifiers, chloride will flow into the cell and drive thetransmembrane potential negative. For inward rectifiers, chloride willbe driven out of the cell and the transmembrane potential will be drivenpositive.

Due to the small difference between the chloride reversal potential andthe resting transmembrane potential, direct stimulation of avoltage-gated chloride channel may result in insufficient transmembranepotential changes. Assays for these ion channels can then be developedusing co-expression and electrical stimulation of a sodium or potassiumcounter-channel in order to produce an inward or outward current.Presence or absence of the chloride current can then be determined bythe absence or presence of a transmembrane potential change when thecounter-channel is electrically stimulated. Preferred cell types includethose cells that express a minimal level of other ion channels, such asCHO-K1, CHL, and LTK (−). The transfection and selection of clonesexpressing ion channels of interest will generally be performed asdescribed above for sodium ion channels normally in the resting state.Alternatively, a cell line which endogenously expresses the channel ofinterest (or the counter-channel) could be used. The labeling andmeasurement of cells with transmembrane potential dyes will generally beperformed as described for sodium ion channels normally in the restingstate.

Typically one would start out with initial conditions using a biphasicsquare wave kernel of 5 msec per phase and amplitude of 25 V/cm. Thekernel would be repeated at a regular rate of about 20 Hz for a totaltrain duration of about three seconds. One would then optimize the pulseamplitude, duration, and then frequency. If necessary changes in thepulse shape could also be explored to determine if these resulted inmore efficient electrical stimulation. The optimal stimulus parameterswill yield the maximum cellular stimulation (compared to cells with thechannel blocked, or not present) with smallest coefficient of variationof the signal among the different test wells, at the lowest electricfield strength, and at the lowest duty cycle for passage of currentthrough the electrodes. After a particular set of parameters is chosen,a titration of staining concentrations for the voltage sensor dye(s)should be performed as described above, to further optimize the signalsize and coefficient of variation of the responses. These procedures(dye concentrations, electric field strength, and stimulus duration andfrequency) can be iterated to further optimize the signal.

During these experiments, the response will be compared for cells withactive channels, and for cells in which the channels arepharmacologically blocked. If a suitable pharmacological agent is notavailable, the blocked state can be emulated with an un-transfected cellline. The optimal stimulus parameters will yield the smallestcoefficient of variation of the difference in signals of the two cellpopulations.

(a)

(b) e) Assay of Ligand Dependent Channels

The ligand-dependent ion channel family is large and diverse.Ligand-dependent ion channels open in response to the binding ofspecific molecules. They typically mediate fast synaptic transmissionbetween neurons, and from neurons to muscle cells. They also mediateslow synaptic transmission and control a variety of regulatorymechanisms. Ligand-gated ion channels are generally onlycharge-selective; that is, they permit the flow of a range of eitheranions or cations but have little specificity. They have enormousvariation in their activation, deactivation, and desensitizationkinetics, all of which can vary from submillisecond to second timeconstants.

When the ligand binds to the receptor of the channel, the channelundergoes one or more conformational changes to activate the channel. Ifthe ligand is removed from the bathing saline, the bound ligandsdissociate and the channel closes. If the ligand remains in the bathingsaline, some channels desensitize by retaining the ligand but movinginto a different conformational state in which the channel is closed.Equilibrium distributions between the activated, deactivated, anddesensitized states vary greatly among channels.

In current assay formats, the transmembrane potential of the cells ismonitored during an addition of ligand. The sudden increase inconductance when the channel opens drives the transmembrane potentialtowards a new reversal potential. Unfortunately, for many ligand-gatedchannels, the new reversal potential is usually within 15 mV of theresting potential. This small change is sufficient to use for signalingwithin cells, but it makes pharmacological assays difficult.

In an electrical stimulation assay for ligand-gated ion channels, oneapproach is to co-express a voltage-gated sodium counter channel withthe ligand gated ion channel of interest. This approach allows us tomodulate the transmembrane potential via electrical stimulation. If thetest compounds are added to the cells during or prior to electricalstimulation, the method enables an analysis of whether the ligand gatedchannel is open or closed. If the ligand-gated channels are open, thehigh resting conductance of the cell will suppress the voltage responseto electrical stimulation. If, however, the ligand-gated channels areblocked, the cells will have a large response to electrical stimulation.The large amount of flexibility in electrical stimulation parametersshould allow us to assay for a large range in resting conductances. Thisis important in the case of ligand-gated channels, because the restingconductance in the presence of ligand is very sensitive to theequilibrium desensitization. Accounting for desensitization andvariations in channel expression, we may have resting membraneresistances ranging anywhere from 10 MΩ to 10 GΩ. With rat brain type Iasodium channels as the counter channel, we can cover this entire range.It should also be possible to screen for both agonists and antagonists.By choosing stimulation parameters such that the response is half-size,agonists will reduce the response while antagonists will increase it.Better screening windows may be obtained by stimulating at higher(agonist assay) or lower (antagonist assay) frequencies. Note thatmodulators of the channel conductance, open time, desensitization, anddeactivation will all be detected.

Preferred cell types include those cells that express a minimal level ofother ion channels, such as CHO-K1, CHL, and LTK (−). The transfectionand selection of clones expressing ion channels of interest willgenerally be performed as described above for sodium ion channelsnormally in the resting state. Alternatively, a cell line whichendogenously expresses the channel of interest (or the counter-channel)could be used. The labeling and measurement of cells with transmembranepotential dyes will generally be performed as described for sodium ionchannels normally in the resting state.

Typically one would start out with initial conditions using a biphasicsquare wave kernel of 5 msec per phase and amplitude of 25 V/cm. Thekernel would be repeated at a regular rate of about 20 Hz for a totaltrain duration of about three seconds. One would then optimize the pulseamplitude, duration, and then frequency. If necessary changes in thepulse shape could also be explored to determine if these resulted inmore efficient electrical stimulation. The optimal stimulus parameterswill yield the maximum cellular stimulation (compared to cells with theligand-gated channel blocked, or not present) with smallest coefficientof variation of the signal among the different test wells, at the lowestelectric field strength, and at the lowest duty cycle for passage ofcurrent through the electrodes. After a particular set of parameters ischosen, a titration of staining concentrations for the voltage sensordye(s) should be performed as described above, to further optimize thesignal size and coefficient of variation of the responses. Theseprocedures (dye concentrations, electric field strength, and stimulusduration and frequency) can be iterated to further optimize the signal.

During these experiments, the response will be compared for cells withactive channels, and for cells in which the channels arepharmacologically blocked. If a suitable pharmacological agent is notavailable, the blocked state can be emulated with an un-transfected cellline. The optimal stimulus parameters will yield the smallestcoefficient of variation of the difference in signals of the two cellpopulations.

(a)

(c) f) Assay of Passive Channels

Many channels have slow or no voltage-activated conductance changes.Prime examples are the some of the channels implicated in cysticfibrosis, particularly the cystic fibrosis transmembrane regulator(CFTR, a chloride channel), the epithelial sodium channel (ENaC) and 4TM potassium channel family members (Wang et al. Ann. N.Y. Acad. Sci.868: 286-303, 1999). A small molecule which acts as an agonist foreither of these channels would be a candidate for a drug whichalleviates cystic fibrosis. Currently, there is no convenient workablehigh throughput screening method for channels of this type.

The proposed assay format for ion channel targets of this type involvesa cell expressing the leak channel of interest in a cell which alsoexpresses a voltage-dependent sodium channel. The channel of interest iscloned into a cell with a voltage-dependent sodium channel. The presenceof the passive current will suppress the positive response of the sodiumchannel when the cells are stimulated. Blocking the passive channel willrestore the large positive voltage response. Optimization of the balanceof currents will be important in this method. Wild-type CHO cell may beuseful for this purpose, although a cell with larger sodium currents(either endogenous or engineered) would be preferable. If the sodiumcurrent is too small relative to the potassium current, thedose-response curve for the passive channel blocker will be shiftedtowards higher concentrations. For example, in the extreme case wherethe passive current is 100 times larger than the sodium current, 99% ofthe passive channels would have to be blocked in order to get a 50%response from the sodium channels.

Preferred cell types include those cells that express a minimal level ofother ion channels, such as CHO-K1, CHL, and LTK (−). The transfectionand selection of clones expressing ion channels of interest willgenerally be performed as described above for sodium ion channelsnormally in the resting state. Alternatively, a cell line whichendogenously expresses the channel of interest (or the counter-channel)could be used. The labeling and measurement of cells with transmembranepotential dyes will generally be performed as described for sodium ionchannels normally in the resting state.

A preferred stimulation protocol uses a biphasic kernel. In general, aseries of initial experiments are conducted using a biphasic square wavekernel repeated at a regular rate for a fixed train duration. The pulseduration varies from about 1 μs to about 1 s, and more preferably fromabout 100 μs to about 20 ms. The pulse amplitude varies from 0 V/cm toabout 60 V/cm, and more preferably from 10 V/cm to 50 V/cm. Thefrequency of stimulation varies between 0 Hz (i.e. a single pulse) and100 kHz, and more preferably from 0 Hz to about 1 kHz. The pulse trainvaries between 0 s (i.e. a single pulse) and about 100 s, and morepreferably between 0 s and 10 s.

Typically one would start out with initial conditions using a biphasicsquare wave kernel of 5 msec per phase and an amplitude of 25 V/cm. Thekernel would be repeated at a regular rate of about 20 Hz for a totaltrain duration of about three seconds. One would then optimize the pulseamplitude, duration, and then frequency. If necessary changes in thepulse shape could also be explored to determine if these resulted inmore efficient electrical stimulation. The optimal stimulus parameterswill yield the maximum cellular stimulation (compared to cells with theligand-dependent channel blocked, or not present) with smallestcoefficient of variation of the signal among the different test wells,at the lowest electric field strength, and at the lowest duty cycle forpassage of current through the electrodes. After a particular set ofparameters is chosen, a titration of staining concentrations for thevoltage sensor dye(s) should be performed as described above, to furtheroptimize the signal size and coefficient of variation of the responses.These procedures (dye concentrations, electric field strength, andstimulus duration and frequency) can be iterated to further optimize thesignal.

It should be possible to screen for both agonists and antagonists. Bychoosing stimulation parameters such that the response is half-maximal,agonists will reduce the response while antagonists will increase it.Better screening windows may be obtained by stimulating at higher(agonist assay) or lower (antagonist assay) frequencies.

During these experiments, the response will be compared for cells withactive channels, and for cells in which the channels arepharmacologically blocked. If a suitable pharmacological agent is notavailable, the blocked state can be emulated with an un-transfected cellline. The optimal stimulus parameters will yield the smallestcoefficient of variation of the difference in signals of the two cellpopulations.

The present invention also includes methods for the quantitativedetermination of cellular and ion channel parameters, and for thequantification of the pharmacological effects of test compounds on theseparameters using electrical stimulation.

b. b) Quantitative Measurements of Membrane Resistances

After the electrical stimulus ends, the cell transmembrane potentialrelaxes to a new resting potential. In the case of voltage-dependentchannel assays, the channels will generally close or inactivate, and thefinal resting equilibrium potential will be the same as before thestimulus. In most cases, the charge built up on the membrane capacitancewill dissipate exponentially through the membrane resistance. Themembrane time constant is simply the product of the membrane capacitanceand the membrane resistance, τ_(m)=R_(m)C_(m). It can be readilydetermined by measuring the membrane capacitance and the membrane timeconstant.

The average membrane capacitance for cells commonly used in these assaysis independent of the exogenous channel, and can easily be measured bypatch clamp methods. The membrane time constant can be readily measuredby measuring the rate of decay of the transmembrane potential andfitting this data to an exponential decay function. Thus by dividing themembrane time constant by the average membrane capacitance for the givencell type, we can quantitatively determine the resting or leak membraneresistance.

A similar analysis can be made to quantitatively measure the membraneresistance while a voltage-dependent channel is open. During theelectrical stimulation, the transmembrane potential will also relaxapproximately exponentially towards a new equilibrium potential. Thus,the membrane time constant of the voltage change at the beginning of thestimulus constitutes a measurement of the time-averaged membraneresistance. Using appropriate scaling factors to account for thefraction of the time that the channel is actually open, we can make aquantitative estimate of the open-channel membrane resistance.

c) Measurement of Release From Inactivation Time Constant

Opening an inactivation ion channel requires holding the transmembranepotential below a threshold for a time on the order of severalmilliseconds. This release from inactivation has important physiologicalimplications. For example, release from inactivation forces a refractoryperiod which prevents back-propagation of action potentials, and limitsthe maximum firing rates of neurons. Pharmacological manipulation ofthis property may be therapeutically relevant

Using repetitive electrical stimulation, we can estimate the averagerelease from inactivation time. This can be done by using electric fieldpulses of variable width. When the pulse width falls below the releasefrom inactivation time, fewer channels will be activated and thetransmembrane potential rise in response to the stimulation will drop.

d. d) Measurement of the Open Channel Time

The open channel time τ_(open) is a function of the inactivationproperties of the channel. We can detect pharmacological manipulation ofthis parameter in a medium-to high-throughput mode by stimulating atvery high frequency. For example, consider an assay for avoltage-dependent sodium channel using the multiple stimulus method.With a fixed monophasic square wave stimulus kernel repeated at a steadyrate, the voltage response increases as the stimulus repetition rateincreases. This is because the sodium channel spends relatively moretime open at higher frequency. However, if the inter-pulse intervalbecomes shorter than the open channel time, the activated sodiumchannels will be driven negative, and thereby deactivated, by thesubsequent stimulus pulse. The stimulation burst frequency at which theresponse flattens is related to the open channel time.

f. e) Electrical Stimulation as an Extracellular Current Clamp Device

In whole-cell recording, current clamp is a mode in which commandcurrents can be driven into the cell while recording the transmembranepotential. Although patch-clamp recording is extremely precise, it is avery low-throughput technique. At an absolute maximum under perfectconditions, a highly trained scientist could determine cellularparameters at a rate of about ten cells per hour. Often, the level ofdetail obtained with the patch-clamp technique is not necessary for drugscreening, but there is currently no method for exchanging detail forspeed. High speed is absolutely crucial for screening large compoundlibraries.

The electric field stimulation techniques discussed herein permit a newtype of current-clamp electrophysiology which we call extracellularcurrent clamp. Voltage-dependent channels can be used to drive commandcurrents into cell cultures, allowing determination of several cellularand channel properties. Extracellular current clamp has a very highthroughput, so that it will be possible to obtain high informationcontent of the pharmacological effects of compound libraries againstspecific ion channel targets. The pharmacology and physiology of achannel can be studied directly, or the channel can be used as a currentgenerator for the study of the cell membrane itself or a second ionchannel.

While the ultimate precision of the microscopic parameters obtainablewith the extracellular current clamp cannot yet approach the patch-clampmethod, we now have the ability to exchange information content forthroughput. That is, the degree of precision at which to makemeasurements can be arbitrarily set. With a single set of stimulusparameters, large libraries can be screened for potential interestingcompounds. A medium throughput secondary screen using a titration ofcompound concentrations can be performed on the hits to determinepotency and specificity. Finally, we can determine such therapeuticallyrelevant properties such as use-dependence and mechanism of action byvarying the stimulus parameters in the presence of the compounds. Atevery stage, the measurements are automatically averaged over manycells, greatly reducing uncertainties associated with cell-to-cellvariability.

There are at least two additional advantages of the extracellularcurrent clamp as compared to patch-clamp analysis. First, the integrityof the cell membrane is not altered during electric field stimulation.The intracellular fluid is completely replaced with pipette solutionduring whole-cell patch clamp recording. Many proteins within the cell,including ion channels, are extremely sensitive to modulators,intracellular messengers, and the ionic environment. The components ofthe cytoplasm are only crudely known, so the soluble components in theintracellular space are always altered.

Therefore, the ‘normal’ physiological state of the cell is onlyapproximated during whole-cell patch clamp analysis, but remains intactwhen using extracellular current clamp.

Second, most cells experience dramatic alterations in gene expressionand behavior when in contact with other cells. Because most cells alsomake gap junctional connections with neighboring cells, whole-cell patchclamp analysis is only reliable when cells are completely isolated fromeach other. Extracellular current clamp can be used on cellsindependently of their degree of confluence, so the cells may be morephysiologically relevant. We can use extracellular current clamp to findout if there are any effects of cell-cell contact on channelelectrophysiology. Then, in conjunction with gene expression analysis,we can relate these changes to regulatory components of the cell.

g. f) Electrical Stimulation as an Extracellular Voltage Clamp Device

In voltage-clamp, the transmembrane potential of the cell is controlledwhile monitoring the current flow. Voltage clamp is generally achievedby adding a feedback loop to a current clamp circuit. In the case of thewhole-cell method, this can easily be achieved with the use of twopipettes simultaneously attached to the same cell. One pipette passes acommand current, while the other senses the voltage. A feedback circuitcompares the measured voltage with the command voltage, and adjusts thecommand current accordingly. Generally, because the cell membraneresistance is large compared to the access resistance of the pipette,the same pipette can be used to command the current and measure thevoltage. Compared to current clamp, voltage clamp is generally a morepowerful method for electrophysiological analysis. Ion channels areextremely sensitive to transmembrane potential, so that analysis of datais far more straightforward when dealing with current measurements at afixed voltage.

Extracellular current clamp can be converted to a voltage clamp methodby adding a feedback loop between the voltage measurement (thefluorescence of the sensor dye) and the current generator (the stimulusparameters). In this case, a transmembrane potential dye with sufficientspeed is required. The dye combination CC2-DMPE/DiSBAC₆(3) has asubmillisecond time constant and should be sufficiently fast to captureall but the fastest cellular events. Based upon the difference of thecommand voltage and the transmembrane potential measurements, a computerwill alter the stimulus parameters. The stimulus parameters are relatedto the current driven into the cell, so we can determine the time courseof the current as a function of the command voltage. This method shouldprove useful in determining the mechanism of action of pharmacologicalagents upon ion channels targets.

h. g) Assays for Intracellular Compartments

The stimulation methods described herein can also be used to modulatethe transmembrane potentials of intracellular organelles that havephospholipid membranes, including the mitochondria and the nucleus. Thiscan be accomplished by first increasing the conductance of the plasmamembrane either by electropermeablization or through the addition ofionophores such as valinomycin or gramicidin A. Then, the intracellularspace is no longer insulated from the applied electric field. Thisallows an electric field applied to the saline to generate transmembranepotential changes across the membranes of intracellular organelles.Then, by staining the cells with dyes which are sensitive to the ionconcentration or transmembrane potential, and which are targeted only tothe specific organelle membrane of interest, the methods presentedherein can be used to modulate and assay the ion channels of theseorganelles. Targeting can be achieved, for example via the use of anaturally fluorescent protein containing suitable subcellular locationsignals as are known in the art.

2. IX Introduction of Exogenous Molecules

Dielectric breakdown of mammalian cell membranes occurs if the electricpotential across the membrane exceeds about 200 mV (Teissie and Rols,1993, Biophys. J. 65:409-413). When the membrane breaks down, pores areformed through the membrane, bridging the intracellular andextracellular spaces. The number and size of the pores increases withincreasing transmembrane potentials (Kinoshita and Tsong, 1977, Nature268:438-441). Increasing the electric field strength above about 60 V/cmon typical mammalian cell lines can electropermeablize the cells. Atrelatively low fields, small pores are created in the cell membranewhich apparently are large enough to admit small ions, but not largeenough to admit molecules as large as DNA (Tsong, 1991, Biophys. J.60:297-306). These pores totally depolarize the cell, driving thetransmembrane potential to near zero. By electropermeablizing cells andmonitoring the transmembrane potential change with a voltage-sensitivedye, the present invention can be used to determine the restingtransmembrane potential of a cell. This will be useful for determiningpharmacological interactions with cells or ion channels, either as aprimary or a secondary screen. For example, in a compound screen againsta voltage-dependent sodium channel, one could perform a multiplestimulus protocol to determine channel activity. Then, by following witha permeablizing protocol, one could determine whether or not the cellmembrane had a normal resting potential in the presence of the compound.

Additionally, using a highly polarized cell line such as RBL cells,voltage sensitive dyes could be easily calibrated byelectropermeablization. The starting transmembrane potential undervarious conditions (for example, various concentrations of extracellularpotassium), and the final transmembrane potential afterelectropermeablization is zero.

Additionally, the size of the pores created by electropermeablizationincreases as a function of the applied electric field. Below 50 V/cm, nopores are created. Between about 60 V/cm and 100 V/cm, pores largeenough to admit monovalent ions are created. Above around 600 V/cm,pores large enough to admit DNA are created (Tsong, 1991, Biophys. J.60:297-306). Thus, this invention can be used to create pores of definedsize in the cell membranes, in a high-throughput manner. This could beuseful for many applications, including delivery to the intracellularspace of impermeant ions, impermeant test compounds or other modulators,DNA or RNA for the purpose of transient or stable transfection, andfluorescent or other indicator dyes.

3. X. Drug Discovery and Screening

a) Drug Screening

The present invention provides for the reliable detection of testcompounds that modulate ion channel function that is significantly moreversatile and robust than previous assay systems. Importantly, thepresent invention provides the ability to modulate the transmembranepotential in intact cells without the requirement of pharmacologicalagents, or membrane destruction, and loss of intracellular contents, asin patching clamping. By providing the ability to externally modulatethe transmembrane potential of living cells, the present inventionenables a wide variety of ion channels to be assayed.

Furthermore, this ability to modulate precisely the voltage dependentstate of an ion channel, has important advantages for drug discoverywhere it provides the opportunity to screen for compounds that interactpreferentially with one state, (i.e. use-dependent blockers). Forexample, several known therapeutically useful drugs (includinganti-arrhythmics, anti-convulsants, and local anesthetics) are known tofunction as use-dependent blockers of voltage-dependent sodium and/orcalcium channels. In each case, total blockade of the targeted channelwould typically result in death. Certain conditions, such as chronicpain, arrhythmia, and convulsions occur when cells become over-active.These conditions can be alleviated or eliminated by blocking thechannels if they begin to open too often. Compounds that are capable ofblocking the channel, but which bind preferentially to the activated orinactivated states(s) rather than the resting state(s), can reduce theexcitability of muscle and neurons. These drugs are effective becausethey do not affect the channel under normal circumstances, but block itonly when necessary to prevent hyper-excitability. However existingmethods of analysis that are compatible with high throughput screeningdo not provide the ability to routinely control the activation state ofthe ion channel in real time.

In particular, the present invention provides for a method for screeningthe effect of a test compound on an ion channel in a defined functionalstate within a cell. The method involves modulating the transmembranepotential of the cell via the use of repetitive electrical stimulationto cycle the ion channel of interest through its activation cycle and toset the transmembrane potential to a desired level suitable for aspecific activation state, or transition between states. Then, during orafter this process a test compound is added to the cell, and thetransmembrane potential is measured.

Typically the results obtained in the presence of the test compound willbe compared to a control sample incubated in the absence of the testcompound. Control measurements are usually performed with a samplecontaining all components and under the same stimulation conditions, asfor the test sample except for the putative drug. Additional controlstudies can be carried out with the ion channel in another voltagedependent state to specifically identify state specific test compounds.Detection of a change in transmembrane potential in the presence of thetest agent relative to the control indicates that the test agent isactive and specific on the ion channel in that state, or during thetransition from one state to another.

Transmembrane potentials can be also be determined in the presence orabsence of a pharmacologic agent of known activity (i.e., a standardagent) or putative activity (i.e., a test agent). A difference intransmembrane potentials as detected by the methods disclosed hereinallows one to compare the activity of the test agent to that of thestandard agent. It will be recognized that many combinations andpermutations of drug screening protocols are known to one of skill inthe art and they may be readily adapted to use with the presentinventions disclosed herein to identify compounds, which affect ionchannels and or transmembrane potentials. Use of the present inventionsin combination with all such methods are contemplated by this invention.

In another aspect the present invention includes the use of a second ionchannel in conjunction with electrical stimulation methods describedherein to set the resting, or stimulated transmembrane potential to apredefined value thereby providing for the ability to assay a first ionchannel of interest. In one embodiment the second ion channel is avoltage regulated sodium or calcium channel which enables the generationof sustained positive transmembrane potentials. In another embodimentthe second ion channel is a voltage regulated potassium channel,enabling the generation of negative transmembrane potentials. The use ofthese second ion channels enables the electrical stimulation method tobe used to set the transmembrane potential to virtually any predefinedlevel.

Because this assay format involves two ion channels, modulators ofeither channel will affect the voltage response. In this case additionalcontrol studies may be carried out with the parental cell lineexpressing only the second ion channel used to set the transmembranepotential. Compounds that block the first ion channel can then bere-tested separately to find out if they have activity against thesecond ion channel.

Typically the test compounds screened will be present in libraries ofrelated or diverse compounds. The library can have individual membersthat are tested individually or in combination, or the library can be acombination of individual members. Such libraries can have at least twomembers, preferably greater than about 100 members or greater than about1,000 members, more preferably greater than about 10,000 members, andmost preferably greater than about 100,000 or 1,000,000 members.

b) Selectivity and Toxicology of Candidate Modulators

Once identified, candidate modulators can be evaluated for selectivityand toxicological effects using known methods (see, Lu, BasicToxicology, Fundamentals, Target Organs, and Risk Assessment, HemispherePublishing Corp., Washington (1985); U.S. Pat. Nos. 5,196,313 toCulbreth (issued Mar. 23, 1993) and U.S. Pat. No. 5,567,952 to Benet(issued Oct. 22, 1996).

For example primary cell lines, or tissue slices can be used to screenfor the effect of the candidate modulator on the response of the ionchannel of interest in its native physiological context. For example, toscreen for drugs that exhibit specific, and/or selective effects onheart cells it may be preferable to use myocytes or other in vitro cellculture model cell lines. In this case, a primary screen could becompleted in a myocyte derived cell line to identify compounds thateither shorten, prolong or block electrically-induced action potentials.

The secondary screen would then be designed to identify compounds thatexhibit potentially adverse effects on the body. For example, this canbe accomplished by screening for the effects of the candidate drug onelectrically excitable tissues such as heart or neuronal tissues, orimmortalized cell cultures derived from these tissues. These tissuesplay critical roles within an organism and any undesired effect of thecandidate drug on the ability of these tissues to be electricallystimulated would be predicted to create potential serious side effectswhen administered. As a consequence, active compounds that also impairedthe ability of these tissues to function could be eliminated fromconsideration as a drug candidate at an early stage, or have medicinalchemistry performed to reduce the side effects.

Additional toxicological analysis of candidate modulators can beestablished by determining in vitro toxicity towards a cell line, suchas a mammalian (preferably human) cell line. Candidate modulators can betreated with, for example, tissue extracts, such as preparations ofliver, such as microsomal preparations, to determine increased ordecreased toxicological properties of the chemical after beingmetabolized by a whole organism, or via their ability to be degraded viaCytochrome P450 systems as described in commonly owned U.S. patentapplication Ser. No. 09/301,525, filed Apr. 28, 1999, U.S. patentapplication Ser. No. 09/301,395 filed Apr. 28, 1999 and U.S. applicationSer. No. 09/458,927 filed Dec. 10, 1999. The results of these types ofstudies are often predictive of toxicological properties of chemicals inanimals, such as mammals, including humans.

The toxicological activity can be measured using reporter genes that areactivated during toxicological activity or by cell lysis (see WO98/13353, published Apr. 2, 1998). Preferred reporter genes produce afluorescent or luminescent translational product (such as, for example,a Green Fluorescent Protein (see, for example, U.S. Pat. No. 5,625,048to Tsien et al., issued Apr. 29, 1998; U.S. Pat. No. 5,777,079 to Tsienet al., issued Jul. 7, 1998; WO 96/23810 to Tsien, published Aug. 8,1996; WO 97/28261, published Aug. 7, 1997; PCT/US97/12410, filed Jul.16, 1997; PCT/US97/14595, filed Aug. 15, 1997)) or a translationalproduct that can produce a fluorescent or luminescent product (such as,for example, beta-lactamase (see, for example, U.S. Pat. No. 5,741,657to Tsien, issued Apr. 21, 1998, and WO 96/30540, published Oct. 3,1996)), such as an enzymatic degradation product. Cell lysis can bedetected in the present invention as a reduction in a fluorescencesignal from at least one photon-producing agent within a cell in thepresence of at least one photon reducing agent. Such toxicologicaldeterminations can be made using prokaryotic or eukaryotic cells,optionally using toxicological profiling, such as described inPCT/US94/00583, filed Jan. 21, 1994 (WO 94/17208), German Patent No69406772.5-08, issued Nov. 25, 1997; EPC 0680517, issued Nov. 12, 1994;U.S. Pat. No. 5,589,337, issued Dec. 31, 1996; EPO 651825, issued Jan.14, 1998; and U.S. Pat. No. 5,585,232, issued Dec. 17, 1996).

Alternatively, or in addition to these in vitro studies, thebioavailability and toxicological properties of a candidate modulator inan animal model, such as mice, rats, rabbits, or monkeys, can bedetermined using established methods (see, Lu, supra (1985); andCreasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology,Oxford University Press, Oxford (1979), Osweiler, Toxicology, Williamsand Wilkins, Baltimore, Md. (1995), Yang, Toxicology of ChemicalMixtures; Case Studies, Mechanisms, and Novel Approaches, AcademicPress, Inc., San Diego, Calif. (1994), Burrell et al., Toxicology of theImmune System; A Human Approach, Van Nostrand Reinhld, Colo. (1997),Niesink et al., Toxicology; Principles and Applications, CRC Press, BocaRaton, Fla. (1996)). Depending on the toxicity, target organ, tissue,locus, and presumptive mechanism of the candidate modulator, the skilledartisan would not be burdened to determine appropriate doses, LD₅₀values, routes of administration, and regimes that would be appropriateto determine the toxicological properties of the candidate modulator. Inaddition to animal models, human clinical trials can be performedfollowing established procedures, such as those set forth by the UnitedStates Food and Drug Administration (USFDA) or equivalents of othergovernments. These toxicity studies provide the basis for determiningthe therapeutic utility of a candidate modulator in vivo.

c) Efficacy of Candidate Modulators

Efficacy of a candidate modulator can be established using severalart-recognized methods, such as in vitro methods, animal models, orhuman clinical trials (see, Creasey, supra (1979)). Recognized in vitromodels exist for several diseases or conditions. For example, theability of a chemical to extend the life-span of HIV-infected cells invitro is recognized as an acceptable model to identify chemicalsexpected to be efficacious to treat HIV infection or AIDS (see, Dalugeet al., Antimicro. Agents Chemother. 41:1082-1093 (1995)). Furthermore,the ability of cyclosporin A (CsA) to prevent proliferation of T-cellsin vitro has been established as an acceptable model to identifychemicals expected to be efficacious as immunosuppressants (see,Suthanthiran et al., supra, (1996)). For nearly every class oftherapeutic, disease, or condition, an acceptable in vitro or animalmodel is available. Such models exist, for example, forgastro-intestinal disorders, cancers, cardiology, neurobiology, andimmunology. In addition, these in vitro methods can use tissue extracts,such as preparations of liver, such as microsomal preparations, toprovide a reliable indication of the effects of metabolism on thecandidate modulator. Similarly, acceptable animal models may be used toestablish efficacy of chemicals to treat various diseases or conditions.For example, the rabbit knee is an accepted model for testing chemicalsfor efficacy in treating arthritis (see, Shaw and Lacy, J. Bone JointSurg. (Br) 55:197-205 (1973)). Hydrocortisone, which is approved for usein humans to treat arthritis, is efficacious in this model whichconfirms the validity of this model (see, McDonough, Phys. Ther.62:835-839 (1982)). When choosing an appropriate model to determineefficacy of a candidate modulator, the skilled artisan can be guided bythe state of the art to choose an appropriate model, dose, and route ofadministration, regime, and endpoint and as such would not be undulyburdened.

In addition to animal models, human clinical trials can be used todetermine the efficacy of a candidate modulator in humans. The USFDA, orequivalent governmental agencies, have established procedures for suchstudies (see, www.fda.gov).

d) Selectivity of Candidate Modulators

The in vitro and in vivo methods described above also establish theselectivity of a candidate modulator. The present invention provides arapid method of determining the specificity of the candidate modulator.For example, cell lines containing related ion channel family memberscan be used to rapidly profile the selectivity of a test chemical withrespect both to its ability to inhibit related ion channels, and theirrelative ability to modulate different voltage dependent states of theion channels. Such a system provides for the first time the ability torapidly profile large numbers of test chemicals in order tosystematically evaluate in a simple, miniaturized high throughput formatthe ion channel selectivity of a candidate modulator.

e) An Identified Chemical, Modulator, or Therapeutic and Compositions

The invention includes compositions, such as novel chemicals, andtherapeutics identified by at least one method of the present inventionas having activity by the operation of methods, systems or componentsdescribed herein. Novel chemicals, as used herein, do not includechemicals already publicly known in the art as of the filing date ofthis application. Typically, a chemical would be identified as havingactivity from using the invention and then its structure revealed from aproprietary database of chemical structures or determined usinganalytical techniques such as mass spectroscopy.

One embodiment of the invention is a chemical with useful activity,comprising a chemical identified by the method described above. Suchcompositions include small organic molecules, nucleic acids, peptidesand other molecules readily synthesized by techniques available in theart and developed in the future. For example, the followingcombinatorial compounds are suitable for screening: peptoids (PCTPublication No. WO 91/19735, 26 Dec. 1991), encoded peptides (PCTPublication No. WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCTPublication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No.5,288,514), diversomeres such as hydantoins, benzodiazepines anddipeptides (Hobbs DeWitt, S. et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer.Chem. Soc. 114: 6568 (1992)), nonpeptidal peptidomimetics with aBeta-D-Glucose scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc.114: 9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho, C. Y. et al., Science 261: 1303 (1993)), and/orpeptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem. 59: 658(1994)). See, generally, Gordon, E. M. et al., J. Med Chem. 37: 1385(1994). The contents of all of the aforementioned publications areincorporated herein by reference.

The present invention also encompasses the identified compositions in apharmaceutical composition comprising a pharmaceutically acceptablecarrier prepared for storage and subsequent administration, which have apharmaceutically effective amount of the products disclosed above in apharmaceutically acceptable carrier or diluent. Acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,stabilizers, dyes and even flavoring agents may be provided in thepharmaceutical composition. For example, sodium benzoate, acsorbic acidand esters of p-hydroxybenzoic acid may be added as preservatives. Inaddition, antioxidants and suspending agents may be used.

The compositions of the present invention may be formulated and used astablets, capsules or elixirs for oral administration; suppositories forrectal administration; sterile solutions, suspensions for injectableadministration; and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes), may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize. In practicing the methods of the invention, the productsor compositions can be used alone or in combination with one another, orin combination with other therapeutic or diagnostic agents. Theseproducts can be utilized in vivo, ordinarily in a mammal, preferably ina human, or in vitro. In employing them in vivo, the products orcompositions can be administered to the mammal in a variety of ways,including parenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, nasally or intraperitoneally, employing a varietyof dosage forms. Such methods may also be applied to testing chemicalactivity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage for the products of the present invention can range broadlydepending upon the desired affects and the therapeutic indication.Typically, dosages may be between about 10 μg/kg and 100 mg/kg bodyweight, and preferably between about 100 μg/kg and 10 mg/kg body weight.Administration is preferably oral on a daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g., Fingl et al., in The Pharmacological Basis of Therapeutics, 1975).It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theinvention into dosages suitable for systemic administration is withinthe scope of the invention. With proper choice of carrier and suitablemanufacturing practice, the compositions of the present invention, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, for example, by means of conventional mixing,dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, or synthetic fatty acid esters, such as ethyl oleateor triglycerides, or liposomes. Aqueous injection suspensions maycontain substances that increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents that increasethe solubility of the compounds to allow for the preparation of highlyconcentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. Such formulations can be madeusing methods known in the art (see, for example, U.S. Pat. No.5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorlywater soluble compounds); U.S. Pat. No. 5,707,641 (therapeuticallyactive proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilicagents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S.Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676(particulate medicaments); U.S. Pat. No. 5,654,286 (topicalformulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No.5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987(liquid formulations); U.S. Pat. No. 5,641,515 (controlled releaseformulations) and U.S. Pat. No. 5,601,845 (spheroid formulations).

B. EXAMPLES

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe invention as defined in the claims appended hereto.

1. Example 1 Analysis of the Electrical Field Uniformity of ParallelPlate Electrodes in Standard Round Wells

To analyze the effect of various electrode, and well designs, a seriesof two-dimensional numerical simulations of the electric fields wereproduced using the software analysis package Quickfield™ 4.1, (Student'sVersion, Tera Analysis, http://www.tera-analysis.com). This softwarepackage creates coarse-grained mesh type electrical field intensity mapsby solving Poisson's equation with a finite-element analysis method intwo dimensions. For the purposes of this analysis, the fringing effectsdue to the gap between the bottom of the electrode and the bottom of thewell were ignored, and the voltage drops from the electrodes to thesaline were also assumed to be negligible. The spatial resolution of themodeling is approximately 0.5 mm.

FIG. 7A shows the results of the simulation using 4 mm wide parallelplate electrodes (710) with a 4 mm gap with a standard electricalpotential difference of 2V located in a standard circular 96-well. Inthis figure, the outer circle (700) is the edge of the well, the twovertical lines (710) are the electrodes, and the dashed circle in themiddle (720) is the area of observation. The gray area (740) is the areain which the electric field remains within ±10% of the mean field in thearea of observation. In the white area (730), the field is less than 10%of the mean, and in the black area (750), the field is more than 10%greater than the mean field. Within the area of observation, thestandard deviation of the field strength is 2% of the mean, and thedifference between the maximum and minimum fields is 10% of the mean.Thus, this geometry satisfies the stated requirements for fielduniformity for use in the present invention.

2. Example 2 Analysis of the Electrical Field Uniformity of PinElectrodes in Standard Round Wells

To determine the predicted field uniformity for two 1.0 mm diameterround pin electrodes placed in a standard 6.2 mm diameter well,separated by a distance of 4.0 mm, simulations were completed with thesame conditions and assumptions as described in Example 1.

In FIG. 7B, the outer solid circle (705) is the edge of the well, thetwo smaller circles (715) are the electrodes, and the dashed circle inthe middle is the area of observation. The gray area (745) is the areain which the electric field remains within ±10% of the mean field in thearea of observation. In the white area (735), the field is less than 10%of the mean, and in the black area (755), the field is more than 10%greater than the mean field. Within the area of observation (725), thestandard deviation of the field strength is 15% of the mean, and thedifference between the maximum and minimum fields is 87% of the mean.Thus, this geometry does not create uniform electrical fields and as aconsequence is not suitable for use with the present invention.

3. Example 3 Analysis of the Electrical Field Uniformity of ParallelPlate Electrodes in Square Wells

FIG. 8A shows a simulation of the field profile for two 6 mm wideparallel plate electrodes with a 4 mm gap in a 6.2 mm square well. Inthis figure, the outer square (800) is the edge of the well. The twovertical lines (810) are the electrodes. The dashed circle in the middle(820) is the area of observation. Of particular note is that theelectric field scale for FIG. 8 has been greatly amplified compared toFIG. 7 to provide contrast for the variations in electrical fieldintensity. The gray area (840) is the area in which the electric fieldremains within ±1% of the mean field in the area of observation. In thewhite area (830), the field is less than 1% of the mean. In thissimulation, at no point is the field more than 1% greater than the meanfield. Within the area of observation, the standard deviation of thefield strength is 0.02% of the mean, and the difference between themaximum and minimum fields is 0.12% of the mean. Thus, this geometrygreatly improves the field uniformity.

The results of the simulation indicate that the primary source of fieldnon-uniformity in the parallel plate system shown in FIG. 7A derivesfrom the rounded walls of the well. In a standard well with a circularcross section, the current density will spread out and then contract asit passes from one electrode to the other, and this spreading generatesnon-uniformity. This can be corrected by using multiwell plates withsquare wells.

4. Example 4 Analysis of the Effect of the Addition of InsulatorElements to Mask Off Rounded Areas of the Wells

FIG. 8B shows a simulation of the field profile for two 4 mm wideparallel plate electrodes with a 4 mm gap in a 6.2 mm diameter roundwell using the standard conditions and analysis procedures as describedin Example 1. Insulators are attached to the electrodes to mask off therounded areas of the well between the electrodes, as shown in FIG. 9A.In FIG. 8B, the outer circle (802) is the edge of the well. The twovertical lines (812) are the electrodes. The dashed circle in the middle(822) is the area of observation. The cross-hatched areas (862) areinsulators attached to the electrodes. The gray area (842) is the areain which the electric field remains within ±1% of the mean field in thearea of observation. In the white area (832), the field is less than 1%of the mean. In this simulation, at no point is the field more than 1%greater than the mean field. Within the area of observation, thestandard deviation of the field strength is 0.2% of the mean, and thedifference between the maximum and minimum fields is 1.0% of the mean.Thus, this geometry greatly improves the field uniformity over the casewhere no insulator is used, but not as much as in the case of squarewells.

The results demonstrate that the field uniformity in standard round wellplates can be greatly increased by filling the area outside of the areadefined by the electrodes with an insulating material. In practice inertplastics such as nylon, tetrafluoroethylene, polycarbonate, or any othernon-porous polymer, or glass, could be used as the insulator material,provided that they are relatively stable to aqueous solutions, easilyfabricated and preferably non-fluorescent. The insulator would typicallybe attached to the electrode, and would not obscure any of the areadefined by the electrodes.

5. Example 5 Analysis of the Effect of Satellite Electrodes on FieldUniformity

To test whether it is possible to compensate for the loss of currentinto the curved edge of the well via the use of satellite electrodes,simulations were carried out at a variety of electrode geometries. FIG.9B shows one possible embodiment of this concept, and FIG. 8C shows theelectric field profile when this geometry is analyzed using Quickfield™as described in Example 1. In this example, two extra pairs of 0.7 mmwide parallel plate electrodes were placed with a 2 mm gap. Theseelectrodes are outside of the area of observation, and are maintained athalf the potentials of their parent electrodes.

In FIG. 8C, the outer solid circle (804) is the edge of the well. Thetwo long solid vertical lines (814) are the parent electrodes, and thefour shorter solid vertical lines (816) are the satellite electrodes.The dashed circle in the middle (824) is the area of observation. Thegray area (844) is the area in which the electric field remains within±1% of the mean field in the area of observation. In the white area(834), the field is less than 1% of the mean, and in the black area(854), the field is more than 1% greater than the mean field. Within thearea of observation, the standard deviation of the field strength is0.2% of the mean, and the difference between the maximum and minimumfields is 1.2% of the mean. Thus, this geometry greatly improves thefield uniformity over the case where no insulator is used, but not asmuch as in the case of square wells. This example demonstrated the useof four satellite electrodes in a specific configuration. By adding moresatellite electrodes outside of the area of observation, and by properlyassigning their potentials as a function of the potentials applied tothe parent electrodes, the electric field uniformity can, in principle,be improved to arbitrary precision.

For example in a round well configuration, field uniformity in thecenter area of observation can be improved by subdividing the parallelplate electrodes into several pieces separated by thin insulatingdividers, as depicted in FIG. 9D. The potential applied to eachelectrode (expressed as a fraction of the potential applied to thecentral most piece) can be individually tuned to maximize the fielduniformity in the area of observation.

This concept can be expanded to allow the use of non-parallel dipperelectrodes, which have several vertical conducting stripes, each ofwhich is independently controlled.

6. Example 6 Analysis of the Effect of the Meniscus on Electrical FieldUniformity

The meniscus created by dipper electrodes when inserted into a wellgenerates variations of saline depth of around 10% across the area ofobservation. This in turn generates variations in the electric field ofaround 10% across the area of observation. These variations exist evenif the electrode design is predicted to create perfect field uniformity.Thus, eliminating the meniscus effect will improve the actual fielduniformity. One possible method to accomplish this is to add aninsulating barrier between the electrodes. FIG. 9C depicts one suchembodiment, wherein the insulating barrier is used to create a flat topsurface for the liquid in the well. The bottom of this barrier, when theelectrodes are inserted into the well, would be designed to sitapproximately 2.5 mm above the bottom of the well. Thus, the barrierwould be partially immersed in saline, and its bottom surface woulddefine the top of the conductive chamber to be flat and perpendicular tothe electrode surfaces. In this way, the electric field would not beperturbed by irregularities in the surfaces of the conductive volume.

7. Example 7 Fabrication of Dipper Electrode Electrical Stimulator

In one embodiment of the electrical stimulator the device is comprisedof a self-locating frame that positions the dipper electrodes into thearray of wells in a 96 well multiwell plate format (FIG. 1). FIG. 1depicts the inserted position of the electrode array. In this example,the electrical stimulation device can be assembled from three functionalparts. The first part is the positioning frame (40) that locates thedevice relative to the plate wells. This frame is made of metal and is asnug fit to the multiwell plate. This frame serves as the locating basefor the second functional part of the system, the retractor mechanism.

The retractor system consists of shoulder bolts (70) and return springs(not visible). The springs are wrapped around the shoulder bolts, andpress against the positioning frame (40) and the bottom of theinsulating cover (90). The return springs hold the electrode assembly inthe retracted position until the electrodes are lowered into the platewells. The retractor mechanism locates the third functional part of thesystem, the electrode array.

The third functional part of the system is the electrode array. Theelectrode array is made up of eight pairs of identical electrode combs(10). The electrode combs are made of stainless steel and are precisionlaser cut to avoid distortion. Each comb has eight tabs of sufficientwidth to nearly span the diameter of the multiwell plate wells. Thebackbone of the comb forms the electrical connection to the tabs (50).Two of these combs form the electrode pairs that are inserted into acolumn of eight wells. The combs are held in position relative to eachother by an insulating precision drilled plate (30) that locates theelectrodes relative to the positioning frame. Insulating spacers (20)maintain electrode separation and index the combs to the drilled plateby means of a pinned interface. A second set of spacers (25) ensuresprecise positioning of the electrodes (10) relative to the plate (30).Alignment shafts (15) are inserted through alignment holes in thespacers (20) and the electrode combs (10) for additional stability. Thecombs and spacers are held in place against the drilled plate by aninsulating cover (90).

The device may be used manually by placing the device on the multiwellplate and pressing down on the electrode assembly to lower theelectrodes into the wells. When the electrodes are fully extended, apair of locking bars (60) is inserted to keep the electrodes extendedinto the wells. Alternatively the electrode array can be automaticallyinserted and retracted in to the wells via standard mechanical orrobotic control systems known in the art.

FIG. 3 shows a block diagram of the major electrical and opticalcomponents. Electrical stimuli were created via a high-power amplifier(320), driven by a pair of digital function generators (380 and 310). Inone embodiment the switch (330) was a National Instruments (Austin Tex.)ER-16 controlled by a National Instruments PC-DIO 24 digitalinput/output card on board the VIPR™ reader controller computer (300).The switch (330) allowed defined wells within a 96-well plate to beelectrically stimulated with any given time protocol. In this case, asingle column of eight wells was stimulated simultaneously. Theamplifier (320) was built using the APEX PA93 chip (Apex MicrotechnologyCorp, Tucson, Ariz.) following a circuit provided by the manufacturer.The amplifier had the following specifications: ±100V DC in, 100 GΩinput impedance, 20×voltage gain, ±90V out, ±3 A out, 10 Ω outputimpedance. The function generators were Tektronix (Beaverton, Oreg.)model AFG310. The first function generator (380) was triggered by theVIPR™ reader software when the stimulus pulse train was required tobegin, and produced a train of TTL pulses to trigger the second functiongenerator (310). The second function generator was programmed with thestimulus waveform kernel.

8. Example 8 Voltage Dependence of Electrical Stimulation

Wild type Chinese hamster ovary cells (CHO cells) endogenously express avoltage-dependent sodium channel and can be conveniently used tovalidate and optimize electrical stimulation parameters. Besides thissodium channel, these cells appear to have gap-junctional connectionsbetween adjacent cells and a very small (˜20 pA) voltage-dependentoutward current.

The voltage dependent sodium channel in these cells (hereafter referredto as NaV1) has electrophysiological characteristics similar to ratbrain type IIa sodium channels. Analysis of the current/voltagecharacteristics of this channel via standard electrophysiology revealsthat typical wild type CHO cells have an average peak current of 100 pAper cell at −20 mV. This corresponds to a membrane resistivity (R_(Na))of about 800 MΩ. Assuming a single-channel conductance of 10 pS, thissuggests that there are only ˜125 sodium channels per cell. In ourhands, CHO cells typically exhibit a resting transmembrane potential(R_(m)) of about −35 mV, a resting membrane resistance >10 GΩ, and acell membrane capacitance (C_(m)) of 15 pF.

To test the voltage dependency of electrical stimulation, wild type CHOcells were seeded into 96 well microtiter plates and incubated in growthmedium for 24-48 hours. They were then rinsed with bath solution 1 andstained for 30 minutes each with 10 μM CC2-DMPE (coumarin), then 3 μMDiSBAC₂(3) (ethyl oxonol as described in Appendix A1). A stimulatorassembly with 96 pairs of stainless steel electrodes (4 mm wide, 4 mmgap) was placed atop the assay plate, as described in Example 7. Theelectrodes were lowered into the saline covering the cells and remained0.5 mm from the bottom of the well. Ratiometric fluorescencemeasurements were made during electrical stimulation using a VIPR™reader as described above, and the data were analyzed according to theprocedures in Appendix A2. At any one time, only one column of eightwells was assayed; the remaining wells received no excitation light orelectrical stimulation. After each plate was assayed, the electrodeswere thoroughly rinsed with distilled water and dried with compressedair, to prevent cross-contamination between plates.

To determine the transmembrane potential changes occurring in the cellsas a result of electrical stimulation, multiwell plates containing thecells were analyzed in a VIPR™ reader. The cells in a 3 mm diameter areaof observation located midway between the electrodes were excited withlight at 400±7.5 nm. The light was generated by a 300 W xenon arc lamp,and passed through a pair of a pair of dielectric interference band-passfilters to select the correct excitation wavelength. Light was directedto and from the cells via a trifurcated fiber optic cable, with onecable for excitation light and two for fluorescence emission.Simultaneous measurements of blue (460±20 nm) and orange (580±30 nm)signals were recorded from each well at 50 Hz, digitized and stored on acomputer. Initial assays were 15 seconds long, and consisted of a 6second stimulation of repetitive (90 Hz repetition rate) biphasic (5ms/phase) square-wave stimulation beginning at 2 seconds at theelectrical amplitudes shown. For two seconds before and seven secondsafter the stimulation burst, no current passed through the electrodes.FIG. 10 shows the ratiometric responses at various field strengths up to32 V/cm. In this case the apparent rise time of the recorded response islimited by the response time of the DiSBAC₂(3) that has a response timeconstant of around 1 second. Below pulse amplitudes of 10 V/cm, noresponse is detectable. Above 20 V/cm, the response is robust andincreases only slightly as the voltage is further increased up to 32V/cm. As shown in FIG. 11, at higher voltages, the peak response(measured after about 5 seconds) shows only further small increases inresponse. The data in FIG. 11 can be fitted to a Boltzman function,which had a midpoint at 18.0 V/cm with a 2.0 V/cm width. The sharpnessof the onset and the flatness of the response at high fields arestrongly suggestive of a threshold phenomenon. The electric field atwhich the response is half maximal (18 V/cm) corresponds toapproximately ±30 mV deviations in transmembrane potential at theextreme edges of the cells, using formulas previously published(Equation 1, see also Tsong, 1991, Biophys. J. 60:297-306; and assumingan average diameter of the cells of 30 μm). It is thereforequantitatively consistent with the stimulation mechanism described abovefor voltage-gated sodium channels normally in the inactivated state.

High intensity electrical fields can result in electroporation of thecell membranes resulting in large relatively non-specific changes intransmembrane potential (Tsong, 1991, Biophys. J. 60:297-306). Toestablish whether or not this is also a major factor in the responses ofthe cells to lower electrical field intensities used here, experimentswere conducted with the sodium channel specific toxin tetrodotoxin(TTX). If the effects of electrical stimulation can be blocked by thetoxin, this would suggest that the effect of electrical stimulation isprimarily mediated by the activation of sodium channels. The results ofthis experiment are shown in FIG. 12. The data was obtained withelectrical field strength of 33 V/cm and demonstrate that TTX was ableto completely block the effect of electrical stimulation with typicalpharmacological characteristics consistent with the blockage of sodiumchannels. The EC₅₀ from the fit to this data is 9 nM, similar to thereported value for TTX in rat brain type IIa (8 nM, West et al., 1992,Neuron 8: 59-70). The fact that this signal is blocked by TTX withnormal pharmacology is strong evidence that the signal generated viaelectrical stimulation is almost entirely due to NaV 1.

9. Example 9 Variation of Cellular Response to Changes in Stimulus PulseWidth and Frequency

To examine the behavior of the cellular response as the stimulus pulsewidth and frequency were varied, experiments were carried out using wildtype CHO cells as described in Example 8 above at a constant fieldstrength of 25 V/cm, while varying the pulse duration and frequency.

The results are displayed in FIG. 13. Each data point represents theaverage of eight wells stimulated at the same time from experimentsderived from five separate plates of wild-type CHO cells. The resultsshow generally that as the frequency of stimulation increases themagnitude of the response increases. One would predict that this effectshould eventually saturate as the transmembrane potential is driven tothe sodium reversal potential (V_(Na)). In this case this does not occurbecause the sodium channel density is too low.

Increasing the pulse duration results in higher relative degrees ofelectrical stimulation at lower stimulation frequencies up to about 10ms, beyond which further increases are less pronounced. Very small pulsedurations (less than 1 ms) also limit the response, apparently becausethe channels are not effectively released from inactivation. Toefficiently induce large cellular responses, the best stimulationparameters are typically in the range in which the pulse duration isgreater than, or equal to the time constant for recovery forinactivation, and sufficiently short so that the frequency ofstimulation is greater than the membrane time constant. Additionally theoptimal frequency of stimulation is typically less than the reciprocalof the average channel open time.

These experiments demonstrate that electrical stimulation can besuccessfully used even in cells that express even relatively low levelsof voltage dependent channels, and can be successfully completed underconditions that do not lead to significant electroporation or celldeath. These experiments also demonstrate methods by which stimuluspulse duration and repetition frequency can be optimized to produceresponses of a desired size.

10. Example 10 Analysis of CHO Cells Expressing an Exogenous SodiumChannel

Chinese hamster ovary cells were stably transfected with a plasmidencoding a voltage dependent sodium channel (hereinafter referred to asNaV2) as described in section VI. Whole-cell patch clamp analysis wasused to confirm the electrophysiological and pharmacological propertiesof this channel prior to analysis via electrical stimulation. The peaktransient sodium current at −20 mV was measured to be 600±300 pA (N=5),with an average cell membrane capacitance of 15±5 pF. The resting cellmembrane resistance was too large to measure accurately (R_(L)>10 GΩ).The resting transmembrane potential was −31±3 mV.

To determine the threshold electric field for stimulation, cells stablyexpressing the sodium channel were plated in 96-well plates and stainedaccording to the protocol in Appendix A1. The electrical stimulationprotocol involved a 20 Hz, 3 second burst of biphasic (5 ms/phase)stimuli with variable field strength using the electrical stimulatordescribed in Example 7.

FIG. 14 shows representative time traces at various field strengths(each curve is the average of eight wells). At low field strengths,there is no detectable cellular response, suggesting that the averagetransmembrane potential changes less than about 1 mV. Between 35 and 90V/cm, the response is stereotyped, with a fixed shape and amplitude.Above 90 V/cm, the peak response stays relatively constant, but theresponse decay time after the stimulus is removed becomes considerablyextended.

Consistent with the experiments shown in Example 8, the response inducedby electrical field strengths up to 85 V/cm could be inhibited by TTXwhereas the response obtained from cells stimulated above 90 V/cm couldnot (data not shown). Therefore we conclude that the fast response isdue to the sodium-channel-opening mechanism outlined above, while theslow response is mainly caused by electropermeablization of the membraneby the electrical field.

This effect is more easily seen by comparing the behavior of the fastresponse (4 seconds after stimulation) and the slow response (tenseconds after stimulation) with increasing field strength. This data isshown in FIG. 15. Fitting the fast response to a Boltzman function, themidpoint of the early response was at E₅₀=26 V/cm, with a width ofΔE=3.5 V/cm. The response was independent of field strength between 40and 80 V/cm, with a slight increase when electropermeablization sets inabove 90 V/cm.

The slower response due to permeablization was first detectable at 90V/cm, and is itself of potential use in some applications. For example,permeablization can be used for resetting the transmembrane potential tozero, or if the permeablization is selective for a specific ion, forresetting the transmembrane potential to the equilibrium value for thation. This could be useful, for example, in an assay for a channel thatsets the transmembrane potential. Examples include potassium andchloride leak channels, potassium inward rectifiers, and low-voltageactivated voltage-gated potassium channels.

These results are consistent with published studies in whichelectropermeablization begins with a threshold transmembrane potentialof around ±200 mV, independent of cell type (Teissie and Rols, 1993,Biophys. J. 65:409-413). Based on formulae reported in that article andwidely accepted in the literature, CHO cells with an average diameter of30 μm will experience ±200 mV transmembrane potential changes whenexposed to a 90 V/cm extracellular electric field.

11. Example 11 Determination of the Effective Release From InactivationTime and the Effective Open-Channel Sodium Conductance

To make quantitative estimates of the effective release frominactivation time and open channel conductance, but without being boundto any specific mechanism of action, the following theory was developedfor experimental verification.

After opening, the sodium channels inactivate with a voltage-dependenttime constant of order 1 millisecond. Because the current passed by theopen sodium channels is strongly voltage- and time-dependent, it is notpossible to easily generate an analytical expression for the voltagechange after a single stimulation. However by making some simplifyingapproximations, we can model average idealized responses to create atestable theory. For the purposes here, we assume that upon opening, thesodium channels behave as a linear conductance above V_(t)=−40 mV with areversal potential at E_(Na)=+60 mV. The conductance g_(Na) isdetermined as the maximal current obtained at −20 mV in a whole-cellpatch clamp experiment. The time dependence of the sodium channelconductance is simplified by assuming that, when the channel activates,it has a fixed conductance g_(Na)=1/R_(Na) for a fixed time τ_(Na)=1.0ms, after which the channel inactivates.

Using a biphasic square wave stimulus kernel (each phase has a time t₁and is repeated at a frequency f=1/T), the total current entering thecell during T is:

$\begin{matrix}\begin{matrix}{I = {C_{m}\frac{\mathbb{d}V}{\mathbb{d}t}}} \\{= \frac{q_{Na} - q_{L}}{T}} \\{= {{\frac{\tau_{Na}}{{TR}_{Na}}\left( {V_{Na} - V} \right)\left( {1 - {\exp\left\lbrack {- \frac{t_{1}}{\tau_{r}}} \right\rbrack}} \right)} + {\frac{1}{R_{L}}{\left( {V_{L} - V} \right).}}}}\end{matrix} & (2)\end{matrix}$

Here, τ_(Na) is the time the sodium channels are open. R_(Na)=1/g_(Na)is the membrane resistance when the sodium channels are open. R_(L) isthe normal (leak) membrane resistance. V_(L) is the leak reversalpotential (i.e. the resting membrane potential). V_(Na) is the sodiumreversal potential. τ_(r) is the time constant for recovery frominactivation; this is actually a function of the hyperpolarizing voltageachieved during the pulse, but here we assume it to be a constant.

In reality, sodium channels from different parts of the cell experiencedifferent membrane potential changes, and the parameters τ_(Na), τ_(r),and R_(Na) have strong dependence upon membrane potential. The fullmodel would take into account the cell morphology, a random distributionof cell orientations, and the potential and time dependence of theseparameters. It would then be possible to convolute these dependencies toproduce effective values for these parameters. These procedures are tooinvolved for the present discussion. We will instead recognize that thevalues that are extracted from fits to these equations representcomplicated averages of the underlying channel properties.

Solving equation (2) for the transmembrane potential change duringstimulation (V−V_(L)) yields:

$\begin{matrix}\begin{matrix}{{\left( {V - V_{L}} \right) = {\left( {V_{Na} - V_{L}} \right){\frac{f}{f_{0} + f}\left\lbrack {1 - {\exp\left( {- \frac{t}{\tau_{rise}}} \right)}} \right\rbrack}}},{where}} \\{f_{0} = {\frac{R_{Na}}{R_{L}{\tau_{Na}\left( {1 - {\exp\left\lbrack {- \frac{t_{1}}{\tau_{r}}} \right\rbrack}} \right)}}\mspace{14mu}{and}}} \\{\tau_{rise} = \left( {\frac{1}{R_{L}C_{m}} + \frac{\tau_{Na}f}{R_{Na}C_{m}}} \right)^{- 1}}\end{matrix} & (3)\end{matrix}$

If the stimulation is carried out for a long enough time such that a newtransmembrane potential is reached, the steady-state equation is:

$\begin{matrix}{\left( {V - V_{L}} \right) = {\left( {V_{Na} - V_{L}} \right){\frac{f}{f_{0} + f}.}}} & (4)\end{matrix}$

To determine the effective release from inactivation time and openchannel conductance, experiments were conducted as described in example8, using a biphasic square wave kernel at a constant amplitude of 43V/cm at varying frequencies and with pulse durations of 20 ms, 10 ms, 5ms, 2 ms and 0.3 ms. The results, shown in FIG. 16, display the responseas a function of stimulation frequency for several pulse durations. Inthis case as predicted, the response saturates at high frequencies asthe transmembrane potential apparently approaches the sodium reversalpotential. To determine the effective release from inactivation time andchannel open time the response R was fitted to the modified Hillequation below.

$\begin{matrix}{R = {1 + \frac{Af}{f + f_{0}}}} & (5)\end{matrix}$

Equation (5) can be derived from equation (4) by recognizing that theratiometric response R=1 for no transmembrane potential change, and islinear in the transmembrane potential change with an uncalibratedproportionality constant A.

In equation (5), A and f₀ are adjustable parameters. The fitting wasperformed using a non-linear least-squares analysis using Origin 6.0software (Microcal, Northampton Mass.).

The parameters T₀=1/f₀ from equation (5) above were extracted from thesefits and plotted against the pulse duration and are shown in FIG. 17.The line in this figure is a fit to an exponential decay, and from thisfit, we extract the release from inactivation time constant (τ_(r))τ_(r)=5.7 ms and R_(L)τ_(Na)/R_(Na)=0.314.

Assuming that τ_(Na)=1 ms and R_(L)=45 GΩ, then R_(Na)=140 MΩ. This inturn means that the peak sodium conductance would be 100 mV/140 MΩ=700pA. This is in excellent agreement with the value measured in whole-cellpatch clamp.

12. Example 12 Analysis of an Exogenous Sodium Channel in a Cell Linewith Other Endogenous Ion Channels

Wild-type HEK-293 cells typically express a variety of endogenouspotassium and chloride currents (Zhu et al., 1998, J. Neurosci. Meth.81:73-83), so that the resting membrane resistance for these cells is5-10 GΩ. As a consequence the membrane time constant for these cells iscorresponding smaller, thus for optimal stimulation of the cells, onewould predict that the electrical stimulation protocol should berepeated at relatively higher frequencies compared to cells withoutendogenous potassium channels in order to generate comparable signals.

To test that a voltage regulated sodium channel could be efficientlyelectrically stimulated using the present invention in this cellularbackground, HEK-293 cells were stably transfected with a voltagedependent sodium channel hereinafter referred to as NaV3. Cells weretransfected and selected as described in section VI and labeled withFRET dyes as described in Example 8. Cells were plated and loaded with15 μM CC2-DMPE and 2 μM DiSBAC₆ (3) and then subjected to a 25 V/cm,biphasic stimulus train repeated at a frequency of 90 Hz and with a 5ms/phase pulse duration. The stimulation pulse train occurred for atotal duration of 3 seconds and the digitization rate for datacollection was 50 Hz.

The response as a function of time (FIG. 18) shows a rapid (<20 ms risetime) initial phase which decays with a time constant of about 40 ms toa stable plateau. A small rebound potential change is also presentbetween the spike and the plateau. We interpret this behavior as due tothe activation of endogenous voltage-dependent potassium channels(K_(V)) that occur after the first stimulus pulse. Activation of theseendogenous potassium channels would be expected to cause a reduction oftransmembrane potential as potassium leaves the cell consistent with theexperimental data. As electrical stimulation continues the transmembranepotential reaches a new equilibrium which is set by the balance ofsodium influx into the cell and potassium efflux out of the cell. At theend of stimulation, the decay time constant of the response is about 143ms, corresponding to a leak resistance of about 9 GΩ.

To determine whether this overall smaller response could be reliablyused for drug discovery were conducted to determine whether the effectsof TTX or tetracaine could be accurately characterized. The resultsshown in FIG. 19 demonstrate that the pharmacological inhibitionprofiles of these drugs using the present invention are consistent withthe known behavior of the NAV3 sodium channel with these agents. Thedose-response curve for TTX could be fitted with a Hill function with anEC₅₀=25 nM and Hill coefficient 1.1. The dose-response curve fortetracaine could be fitted to a curve with an EC₅₀=11 μM and Hillcoefficient 0.97. These results suggest that the response is caused bysodium channel activity and that pharmacological information on knownand unknown compounds can be obtained using this method.

13. Example 13 Analysis of HEK-293 Cells Expressing the NaV4 SodiumChannel

To determine whether the present method is generally applicable to awide range of different sodium channels, HEK-293 cells were stablytransfected with another voltage dependent sodium channel, hereinafterreferred to as NaV4. These cells were transfected, selected and loadedwith FRET dyes as described in section VI and Example 8. The results ofa dose-response curve for tetracaine on this channel are shown in FIG.20. Here the data points are averages and standard deviations of eightwells and the solid line is a fit to a Hill function with an estimatedEC₅₀=35 μM and Hill coefficient 1.35. These results are consistent withthe known pharmacology of this ion channel and demonstrate again thatthe cellular response is caused primarily by sodium channel activity.

14. Example 14 Analysis of HEK-293 Cells Expressing a Mixture ofVoltage-activated Chloride and Potassium Channels

A demonstration of the direct stimulation of voltage-dependent chlorideand potassium channels was performed using wild-type HEK-293 cells,which endogenously express a mixture of several voltage-activatedchloride and potassium channels (Zhu, Zhang et al. 1998). Wild-typecells were grown in 96-well microtiter plates and assayed at confluenceafter staining with the FRET dyes according to the protocol in AppendixA1. Initial stimulus parameters included a 3 second long electricalstimulation at 20 Hz with a biphasic square wave stimulus kernel with apulse duration of about 5 ms/phase. Stimuli were performed at varyingelectric field intensities to determine the threshold field strength fora measurable cellular response, and in the presence or absence ofpotassium channel blockers.

FIG. 21 shows the cellular voltage response obtained during thisexperiment. In this figure, each panel contains the ten-second timetrace of the response for a single well. The panels are laid out tomatch their relative positions on the plate. The vertical axis in eachpanel is the background subtracted, normalized fluorescence ratio of theFRET voltage sensitive dye combination CC2-DMPE/DiSBAC2(3), changes inthis quantity are roughly proportional to changes in the membranepotential. Each column had identical stimulation conditions, withincreasing electric field strength from left to right across the plate.The twelfth column of the 96 well plate (not shown) contained no cellsand were used for background subtraction. Rows 6-8 contained 10 mM TEAto block the voltage dependent potassium channels. At the lowest fieldstrengths tested, there was no detectable response. At intermediateelectrical fields, a negative voltage response can be seen which rapidlydecays when the stimulus is removed. At the highest fields a largepositive response is elicited. This behavior sets in above 50 V/cm,similar to the electropermeablization threshold seen in CHO cellsexpressing NaV1, (Example 8).

FIG. 22 shows the response averaged between 4.5 and 5.0 seconds ofstimulation as a function of the electric field intensity. The largepositive responses above 60 V/cm were excluded to show thechannel-dependent negative responses. The coefficient of variation ofthe response is generally extremely small, yielding exceptionally largescreening windows (see Appendix A3). For the unblocked data for 20-40V/cm, the difference between stimulated and unstimulated wells is over20 standard deviations.

Tetraethylammonium (TEA), a well-known potassium channel blocker (Hille,1992, Ionic Channels of Excitable Membranes), was added to rows 6, 7,and 8 at a fully-blocking concentration of 10 mM. This treatmentpartially blocks the response. This result is consistent with theexistence of both potassium (blocked by TEA) and chloride (unaffected byTEA) channels in these cells that respond to electrical stimulation. Theeffect of the potassium channels can be isolated by blocking thechloride channels with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid(DIDS) or 4-acetamido-4-isothiocyanostilbene-2,2′-disulfonic acid (SITS;see Hille, 1992, Ionic Channels of Excitable Membranes). Then, the samecell line could be used to screen two channel classes.

15. Example 15 Identification of State Dependent Blockers

Any proposed screening system should preferably be able to reproduce thepharmacology of known compounds as determined by accepted methods. Toverify that this was the case for the present invention, a series oftest compounds of defined activity were analyzed using a CHO cell linethat expresses the NaV2 channel. To accomplish this, cells were culturedin 96 well plates and stained with voltage sensitive dyes as describedin Appendix A1. Test compounds were added to the cells with the oxonolloading buffer. Unless otherwise noted, the compounds were tested as inreplicates of 8, with ⅓ dilutions across eleven columns of the assayplate.

FIG. 23 shows the time traces for selected concentrations of the sodiumchannel blockers tetrodotoxin (TTX) and tetracaine.

Tetrodotoxin is a potent, reversible, non-state specific sodium channelantagonist. By comparison tetracaine is a use dependent sodium channelblocker that exhibits different affinities for different sodium channelstates.

The results show that the present invention provides for highlyreproducible results with relatively little variability either betweensamples or between plates. In FIG. 23 the effect of TTX can be seen as aprogressive loss of response, without significant changes in the shapeof the response. By comparison with tetracaine the responses not onlydecreases, but changes shape as the concentration varies. The C.V. forthese experiments were 10% (TTX) and 9% (tetracaine), compared totypical CVs using the same voltage dyes, but traditional liquid additionwere 16% (TTX) and 18% (tetracaine).

Importantly the results also show that the present invention canidentify the state dependent blockage of the sodium channel bytetracaine. The use-dependent block of tetracaine is more apparent inthe dose-response curves shown in FIG. 24. For TTX, the channel block isindependent of the time window used for calculating the response. Fortetracaine, however, the blockade is an order of magnitude stronger at 3seconds than at 1 second. Under the same stimulation conditions, otheruse-dependent blockers (lidocaine and bupivicaine) showed a smalleramount of shift in the dose-response curves. The EC₅₀ values obtained bythe electrical stimulation protocol for lidocaine were similar to thehigh-frequency values reported in the literature (see Table 4); thissuggests that lidocaine and bupivacaine have fast enough use-dependenceto be fully saturated at the 20 Hz stimulus used here. This in turnsuggests that we can explore the use-dependent properties of localanesthetics by varying the stimulation frequency.

Table 4 lists the blocking concentrations for several sodium channelantagonists. The literature values reported have all been measured usingwhole-cell patch clamping, and are thus based on direct measurements ofthe sodium channel current.

TABLE 4 i. Pharmacology of NaV2 in the electrical stimulation assayElectric field Literature ompound stimulation value Reference Tetracaine0.19 Bupivacaine 1.0 Lidocaine 30 11 a 97 d Phenytoin 24 19 a 36 dWIN-17317 0.009 0.009 b (a) etrodotoxin 0.006 0.008 c saxitoxin 0.001 cverapamil 3 d capsaicin 1.6 amiloride >1000 References a Ragsdale etal., 1996, Proceedings of the National Academy of Sciences, U.S.A. 93:9270-9275 b Wanner et al., 1999, Biochemistry 38: 11137-11146. c West etal., 1992, Neuron 8: 59-70. d Ragsdale et al., 1991, MolecularPharmacology 40: 756-65.

In Table 4, the table entries are EC₅₀ values (in micromolar) for fitsto the dose-response curves from each assay. Each experiment was donetwice, with four wells per drug concentration per experiment. In eachexperiment, eleven concentrations were used, spanning five orders ofmagnitude in concentration. Reported values are the averages of thecalculated EC₅₀ from each experiment. In the cases of use-dependentblockers, the lowest recorded values are quoted.

WIN-17317 and TTX are potent tonic blockers of a variety of sodiumchannels. These compounds can be detected using the electricalstimulation format, which yields blocking potencies near the literaturevalues.

The first four drugs (lidocaine, bupivicaine, tetracaine, and phenyloin)are use-dependent blockers. That is, they have different affinities forthe various states of the channel. They are of great therapeuticrelevance, since at the proper concentration, they can block damagingrepetitive bursting of neurons and muscle cells while leaving normal,low-frequency activity unaffected. In all cases, the measured blockingconcentration measured with electric stimulation is close to thereported literature value. The electrical stimulation assay format isthe only reliable high-throughput method for detecting all modulators ofsodium channels, including agonists, antagonists, and use-dependentblockers.

16. Example 16 Applicability for High Throughput Screening

For the purposes of high throughput screening, the responses should bereliable enough to confidently tell the difference between active andinactive compounds. This can be quantified by examining the distributionof the responses obtained under identical stimulation conditions,comparing native channels with fully blocked channels. Due toexperimental uncertainty and noise in the system, there will be somescatter in the responses. We would like to be able to statisticallyquantify this scatter, and use it to predict the probabilities ofmisidentifying responses as either false positives or false negatives.

To do this a plate of cells expressing the NaV2 voltage-dependent sodiumchannel was loaded with the FRET dyes. One well per column was‘randomly’ spiked with 1 μM TTX, approximately 200 times thehalf-blocking concentration. The cells were assayed with a 20 Hz, 3 secburst of 25 V/cm, 5 ms/phase, biphasic stimuli. The results are shown inFIG. 25. The wells spiked with TTX can easily be distinguished by eye asthe wells with little or no detectable response.

The ratiometric response two seconds after the stimulus began is shownin FIG. 26. The two populations (blocked and unblocked) can easily bedistinguished. The average blocked response was 1.011±0.004 while theaverage unblocked response was 2.67±0.21. The coefficient of variationfor the unblocked response is 13%. The screening window (i.e. thedifference between the populations normalized to the standarddeviations, see Appendix A3) is 7.8(σ₁+σ₂), where σ₁=0.21 is thestandard deviation of the unblocked response and σ₂=0.004 is thestandard deviation of the blocked response. If we take the cutoff pointto distinguish blockers from nonblockers midway between the populations(at 1.042), then the rate of statistical false negatives and falsepositives (assuming a normal distribution) is 1−prob(7.75)=10⁻¹⁴. Thissuggests that during a screen of a large compound library (10⁸compounds), the probability of encountering a single false positive orfalse negative during the entire screen is only one in a million. Forcomparison, if the difference between the populations were only 3 andthe cutoff was optimally placed, the false positive/negative rate wouldbe 0.3%, a factor of 10¹¹ higher. For an actual screen, in which wewould want to include as hits compounds which do not give completeblock, a tradeoff exists between detecting weak pharmacological activityand the rate of false positives. If, for example, we desire a falsepositive rate of 0.1%, then in this screen we can put the screeningcutoff at 3.3 standard deviations below the mean of the unblockedresponse, or at 1.97. In this case, the rate of false negatives iseffectively zero, and compounds which block only 50% of the responsewill be identified as hits.

Mathematically, there are two reasons that the blocked and unblockedpopulations overlap so little. First, the coefficient of variation ofthe unblocked response is relatively small. That is, each response isnearly identical to every other response. Second, and perhaps moreimportantly, there is absolutely no detectable response from the blockedwells. The scatter from blocked wells is consequently extremely small,so that we can place the boundary for distinguishing the populationsvery low.

In assays performed using liquid addition protocols for stimulation,addition artifacts generally give some small response with an associatedscatter. The scatter of the blocked response reduces the screeningwindow, increases the probability of false positives and falsenegatives, and limits the screener's ability to identify partialblockers.

17. Example 17 Screening in Complex Cell Lines

The feasibility of electrical stimulation of cells expressing multiplechannels was demonstrated using cultures of the HL5 cell line. Thesecells were generated by immortalizing cardiac muscle cells (Claycomb etal., 1998, PNAS 95: 2979-84). They contain several voltage-activatedsodium, calcium, and potassium channels, as well as a strong inwardrectifier potassium current and potassium and chloride leak currents.Cells were grown in 96-well microtiter plates and assayed at confluence.They were stained according to the protocol in Appendix A1. Ratiometricfluorescence measurements were made during electrical stimulation usingVIPR™ as described above, and the data were analyzed according to theprocedures in Appendix A2. Stimulus parameters were arbitrarily chosento be: 3 second long burst at 10 Hz with a biphasic square wave stimuluskernel with a pulse duration of 5 ms/phase. Stimuli were performed atvarying electric fields to determine the threshold field. Two rows ofcells contained 10 μM TTX to partially block the cardiac sodium channel,and two rows contained 10 mM TEA to block the voltage-dependentpotassium channels. FIG. 27 shows the normalized responses of each well.Generally as the electric field strength increases, the cellularresponse increases. The last three columns show signs ofelectropermeablization as the voltage continues to increase. In columns6, 7, and 8, the ratio actually rebounds below the starting ratio,suggesting an after-hyperpolarization (a phenomenon caused by slowclosing of voltage-dependent potassium channels).

The rate of the cellular response is extremely fast, and may beapparently limited by the ability of the ethyl oxonol to rapidlyredistribute within the membrane. The rapid response is consistent witha high resting conductance of the cell due to the leak currents and theexpression of potassium inward rectifier channels. TTX partially blocksthe positive response, indicating that it is at least partially due tothe voltage-dependent sodium current.

FIG. 28 shows the response of the untreated cells (rows 1-4) as afunction of the applied electric field. The response increasessigmoidally with the electric field. Above 50 V/cm, there is a sustainedsignal which is unaffected by TTX. As discussed previously, thisbehavior is consistent with the electropermeablization of the cellularmembrane at high electric field strengths. Also shown in FIG. 28 is thescreening window (see Appendix A3) as a function of the stimulus field.

These results demonstrate that HL5 cells can be effectively assayedusing the electrical stimulation technique. Compounds which are known tomodify different ion channels cause detectable changes in the response.Because these ion channels are identical to those expressed by theheart, such an assay would be useful as a secondary screen, to eliminateor mark for modification those compounds which may interfere with normalheart function. It could also be useful as a primary screen, to discovercompounds which may have desirable effects on any one (or a combination)of the heart ion channels.

18. Example 18 Electrical Stimulation of Cell Cultures Using SurfaceElectrodes

Surface mounted electrodes were prepared on glass coverslips coated withchromium (as an adhesion layer) and gold (as a conductive layer). Themetallized coverslips were custom-built by Thin Film Devices, Inc.(Anaheim Calif.). The coverslips were one inch square, 0.17 mm thickCorning 7059 glass. Metallization was performed by vacuum sputteringdeposition. The chromium layer was approximately 1000 Å thick, andserved as an adhesion layer. The gold layer was approximately 5000 Åthick, and served as a conductive layer. The resistivity of thedeposited metal was less than 0.1 Ω/square. A 4-mm gap was etchedthrough the metal by hand-masking the metal surface with achemically-resistant polymer (S1400-27, Shipley Co., Marlborough Mass.),then etching through the metal layers with five minutes in Gold EtchantTFA, followed by five minutes in Chromium Etchant TFD (Transene Co.,Danvers Mass.). The coverslips were attached to the bottoms of 96 wellplates with silicone elastomer (Sylgard 184 (Corning), cured 90 minutesat 70° C.). After sterilizing with 365 nm UV irradiation for 30 minutesand coating with the cell adhesion molecule poly-D-lysine (molecularweight 300,000, 1 mg/mL in Dulbecco's phosphate buffered saline for 30minutes, then rinsed 3 times with distilled water), living cells couldbe successfully grown and cultured on the electrode surfaces.

To validate the surface electrode stimulator CHO cells at an initialdensity of approximately 1000 cells/mm were plated into the wells of the96 well plate and left to attach for approximately 16 hours. These cellswere transfected to express a potassium channel, which set thetransmembrane potential to around −80 mV, and the NaV3 sodium channel.After reaching confluence, the cells were loaded with thevoltage-sensitive FRET dye combination of CC2-DMPE and DiSBAC₂ (3) asdescribed in Appendix A1. The metal surface electrodes were connected tothe output of a pulse generator, which in this case was anexponential-decay electroporator (Gene Pulser II, Bio-Rad Corp.,Hercules Calif.). Ratiometric fluorescence imaging was performed on aZeiss Axiovert TV microscope, equipped with a 75 W xenon arc lamp lightsource. Excitation light was filtered using a 405±10 nm dielectricinterference filter and a 445 DXCR dichroic mirror. Emission light wassplit with a second 525XR dichroic mirror, and measured with a pair ofHamamatsu HC124 photomultiplier tubes (PMTs). One PMT had a 475±40 nmdielectric interference filter in front of it to monitor the bluefluorescent signal. The second PMT had a 580±35 nm dielectricinterference filter in front of it to monitor the orange fluorescentsignal. The optical filters and dichroic mirrors were purchased fromChroma Technology Corp., Battleboro Vt. Ratiometric fluorescence imagingwas performed on fields containing approximately 100 cells. Correctionfor background fluorescence was performed by measuring the blue andorange signals in a field with no cells, then subtracting these from thesignals obtained from the cells. Then the ratiometric signal,proportional to the transmembrane potential changes, was calculated asdescribed in Appendix A2.

The stimulation protocol used single, monophasic electric field pulsesof variable amplitude. The pulses were exponential-decay waveforms witha 4.3 ms decay time constant. The amplitude at the beginning of thepulse was varied from zero to 56 V/cm.

A typical voltage response for CHO cells expressing a potassium channeland the NaV3 sodium channel after a three separate 45 V/cm stimulationresponses are shown in

FIG. 29 for the same field of cells, demonstrating repeatability of theresponse. The speed of the response in this case is limited primarily bythe response time of the mobile hydrophobic dye, which for the ethyloxonol used is about 0.5 second.

The average ratiometric response of a population of cells grown in a 96well multiwell plate stimulated with monophasic stimuli of varying fieldstrengths is shown in FIG. 30. The points in this curve are the averagepeak response of 4 stimulations on the same culture. As is to beexpected from an action-potential-type response curve, there is nodetectable response below about 18 V/cm. The threshold region isrelatively narrow. Between about 20 and 40 V/cm the response increaseswith increasing field strength. Above 40 V/cm the response plateaus.

19. Example 19 Analysis of Wild-type RBL Cells Expressing IRK1

Rat basophilic leukemia (RBL) cells endogenously express the potassiuminward rectifier channel IRK1 (Wischmeyer et al, Pflugers Arch.429:809-819, 1995). This channel selectively conducts potassium ions,with a highly non-linear conductance characteristic. The conductance isnearly linear below the potassium reversal potential V_(K), and rapidlydrops to near zero beginning at about 10 mV positive of V_(K). Cellsexpressing large amounts of inward rectifier channels tend to haveresting transmembrane potentials within a few millivolts of V_(K).

On the side of the cell where the transmembrane potential is drivenpositive by an external electric field applied to cells expressing IRK1and few other ion channels, the IRK1 channels will rapidly close andcease conducting. On the side of the cell where the transmembranepotential is driven negative, the IRK1 channels will open and passpotassium current. If this side of the cell is driven sufficientlynegative, so that the local transmembrane potential is more negativethan V_(K), a net inward potassium current will exist. This current willcause a positive global transmembrane potential change. Because the IRK1channel does not inactivate, this current should be sustained for aslong as the external field is applied.

Adherent RBL cells were seeded into 96-well plates and loaded with FRETdyes as described in Appendix A1. Three rows of wells contained 400 μMbarium chloride to block the IRK1 channel. The plates were analyzedusing a VIPR™ reader while being electrically stimulated with a biphasicstimulus train repeated at a frequency of 50 Hz and with a 5 ms/phasepulse duration. The stimulation pulse train occurred for a totalduration of 5 seconds and the digitization rate for data collection was50 Hz. The applied electric field was fixed for each column of eightwells, and was varied from 7.2 to 72 V/cm. The data were analyzedaccording to the procedures in Appendix A2. The normalized ratio afterthree seconds of stimulation was calculated, averaged for the twopopulation of wells (with and without barium block), and plotted as afunction of the applied field in FIG. 31. The error bars are standarddeviations of the responses. Open squares are the responses withoutbarium block; solid circles are the responses with barium block. Thedata from the wells with barium block indicate that there is nodetectable voltage change during stimulation until the field reaches 80V/cm, at which point some electropermeablization may be occurring. Theunblocked wells show nearly linear response above a threshold at around20 V/cm. This example clearly shows that the present invention can beused to modulate the transmembrane potential in either positive ornegative directions, depending upon the stimulus parameters and theproperties of the ion channels expressed by the cell.

The present invention expands the applicability of electricalstimulation to include non-excitable cells, by providing instrumentationand methods that enable effective stepwise control of membrane potentialwithout resulting in significant electroporation. The present inventionachieves this result via the use of highly uniform, repetitive pulses ofelectrical stimulation applied to the medium surrounding the cells. Theapplied electric fields typically do not directly alter the averagetransmembrane potential of the cell, but instead create symmetricpositive and negative transmembrane potential changes on the sides ofthe cell facing the cathode and the anode, respectively.

The approach exploits the ion selectivity and the non-linear gating andconductance characteristics of voltage-dependent ion channels. Theapproach also exploits the fact that typical intact cells have long timeconstants for decay of transmembrane potential changes. Even in thosecases where the charge injected into the cell by a single stimulus pulseis too small to be detected reliably, appropriately applied multiplestimulus pulses can build large net transmembrane potential excursions.By varying the number, duration, and the shape and amplitude of thepulses, it is possible to artificially set, or change the transmembranepotential of living cells in a fashion that is similar to patchclamping. Other channels, leak currents or transporters that are notclassically considered voltage-dependent, can also be assayed byinducing transmembrane potential changes using a second,voltage-dependent channel and detecting the current flow ortransmembrane potential changes as a result of activation of the targetchannel or transporter.

The present method is robust, compatible with optical detectionmethodologies and readily amendable to a wide range of potentialapplications including high throughput screening for use in drugdiscovery. In many assay formats direct electrical stimulation avoidsthe requirement for liquid addition, making the assay simpler. Complexmanipulations of the transmembrane potential can readily be accomplishedusing variations in the stimulation protocol. Thus, virtually anyvoltage-sensitive channel can be induced to open regardless of the stateof inactivation or voltage dependency. For high throughput drugdiscovery this relaxes the requirements for specialized cell types, andallows assays to be rapidly performed with readily available cell lines.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

V. APPENDICES

A1. Staining Protocol of Voltage FRET Dyes

1. Reagents

140 mM NaCl (a) Assay buffer #1 4.5 mM KCl 2 mM CaCl₂ 1 mM MgCl₂ 10 mMHEPES 10 mM glucose pH 7.40, 330 mOs/kg 100 mg/mL pluronic 127 in dryDMSO (b) Pluronic stock (1000X) 10 mM DiSBAC₂(3) in dry DMSO (c) Oxonolstock (3333X) 10 mM CC2-DMPE in dry DMSO (d) Coumarin stock (1000X) 200mM ESS-CY4 in water (e) ESS-CY4 stock (400X)

2. Loading and Assay Protocol

-   1. Preparation of CC2-DMPE loading buffer. Normally for a 96-well    plate, 10 mL of staining solution will be prepared per plate.    -   i) Mix equal volumes (10 μL) of coumarin stock and pluronic        stock in a tube.    -   ii) Add 10 mL Assay Buffer #1 to tube while gently vortexing.        -   Loading concentration: 10 μM CC2-DMPE and 0.1 μg/ml            pluronic.-   2. Prepare oxonol loading buffer:    -   i) Mix equal volumes (3.3 μL) of oxonol stock and pluronic stock        in a tube.    -   ii) Add 10 mL Assay Buffer #1 to tube while gently vortexing.    -   iii) Add 25 μL ESS-CY4 while vortexing.        -   Loading concentration: 3 μM DiSBAC₂(3), 0.2 μg/ml pluronic,            and 0.5 mM ESS-CY4.    -   iv) If required, combine test compounds with the loading buffer        at this time.-   3. Rinse cells twice with Assay Buffer #1, removing all fluid from    wells each time.-   4. Add 100 μL CC2-DMPE loading buffer to each well. Incubate 30    minutes at room temperature, avoiding bright light.-   5. Rinse cells twice with Assay Buffer #1, removing all fluid from    wells each time.-   6. Add 100 μL oxonol loading buffer to each well.-   7. Incubate for 30 minutes at room temperature avoiding bright    light. Use immediately.    A2. Analysis of VIPR™ Reader Data

Data were analyzed and reported as normalized ratios of intensitiesmeasured in the 460 nm and 580 nm channels. The process of calculatingthese ratios was performed as follows. On all plates, column 12contained Assay Buffer #1 with the same DiSBAC2(3) and ESS-CY4concentrations as used in the cell plates, however no cells wereincluded in column 12. Intensity values at each wavelength were averagedin initial (before the stimulus) and final (during the stimulus)windows. These average values were subtracted from intensity valuesaveraged over the same time periods in all assay wells. The ratiosobtained from samples in the initial (Ri) and final windows (Rf) aredefined as:

$\begin{matrix}{{Ri} = \frac{\left( {{{intensity}\mspace{14mu} 460\mspace{14mu}{nm}},{{initial} - {{background}\mspace{14mu} 460\mspace{14mu}{nm}}},{initial}} \right)}{\left( {{{intensity}\mspace{14mu} 580\mspace{14mu}{nm}},{{initial} - {{background}\mspace{14mu} 580\mspace{14mu}{nm}}},{initial}} \right)}} & \left( {{A2}{.1}} \right) \\{{Rf} = \frac{\left( {{{intensity}\mspace{14mu} 460\mspace{14mu}{nm}},{{final} - {{background}\mspace{14mu} 460\mspace{14mu}{nm}}},{final}} \right)}{\left( {{{intensity}\mspace{14mu} 580\mspace{14mu}{nm}},{{final} - {{background}\mspace{14mu} 580\mspace{14mu}{nm}}},{final}} \right)}} & \left( {{A2}{.2}} \right)\end{matrix}$

Final dada are normalized to the starting ratio of each well andreported as Rf/Ri.

A3. Screening Window

The screening window W for a response is defined as follows. Data frommultiple wells at identical stimulus conditions are required. Thecontrol wells can either be pharmacologically blocked or untransfectedcell stimulated with the full electric field. Alternatively, one mightuse transfected cells with no stimulus applied.

Responses from experimental and control wells are measured. The averageand standard deviations of the responses in the experimental (R±ΔR) andcontrol (C±ΔC) wells are calculated. The screening window is defined asthe difference between experimental and control signals normalized tothe sum of the standard deviations.

$\begin{matrix}{W = \frac{R - C}{{\Delta R} + {\Delta C}}} & \left( {{A3}{.1}} \right)\end{matrix}$

A general rule of thumb for an acceptable screening window is W>3. Thisallows one to choose a cutoff line midway between control andexperimental responses which ensures a false negative/positive rate lessthan 1%. Assuming a normal distribution, the false positive/negativerate as a function of the screening window W is:

$\begin{matrix}\begin{matrix}{P_{false} = {1 - {{prob}(W)}}} \\{= {1 - {\frac{1}{\sqrt{2\pi}}{\int_{W}^{W}{{\exp\left( {- \frac{t^{2}}{2}} \right)}\ {\mathbb{d}t}}}}}}\end{matrix} & \left( {{A3}{.2}} \right)\end{matrix}$

Table A3.1. The false positive/negative rate P(W) as a function of thescreening window W as defined in Equation A3.1. This calculation assumesthat the cutoff for identification of a hit is placed an equal number ofstandard deviations away from the positive and negative controlresponses.

TABLE A3.1 The false positive/negative rate P(W) as a function of thescreening window W as defined in Equation A3.1. This calculation assumesthat the cutoff for identification of a hit is placed an equal number ofstandard deviations away from the positive and negative controlresponses. W P(W) 1 0.3173 2 0.0455 3 0.0027 4 6.334E-5 5 5.733E-7 61.973E-9 7 2.559E-12 8 1.221E-15 9   <1E-18 10   <1E-18

1. A method of screening a plurality of drug candidate compounds againsta target ion channel comprising: expressing said target ion channel in apopulation of host cells; placing a plurality of said host cells intoeach of a plurality of sample wells; adding a candidate drug compound toat least one of said plurality of sample wells; affecting the target ionchannel by modulating a transmembrane potential of said host cells insaid at least one well with an application of a series of electric fieldpulses applied with extracellular electrodes extending down into said atleast one well so as to set said transmembrane potential to a levelsuitable for a specific ion channel activation state or transitionbetween states, wherein the frequency of the electric field pulses (f)is within the range τ_(M) ⁻¹≦f≦τ_(b) ⁻¹ where τ_(M) is a time constantfor decay of transmembrane potential changes, and τ_(b) is an averagetarget ion channel open time, wherein the pulses at said frequency causea sustained transmembrane potential change via a stepwise accumulationor loss of ions over the course of said series of pulses; and detectingtransmembrane potential characteristics of said plurality of cells overan area of observation in said at least one well to detect an effect ofsaid candidate drug compound on said target ion channel.
 2. The methodof claim 1, additionally comprising selecting a host cell line having anormal resting transmembrane potential corresponding to a secondpre-selected voltage dependent state of said target ion channel.
 3. Themethod of claim 1, wherein said electric fields are biphasic.
 4. Themethod of claim 1, wherein electric fields cause said target ion channelto cycle between different voltage dependent states.
 5. The method ofclaim 1, wherein said electric fields cause said target ion channel toopen.
 6. The method of claim 1, wherein said electric fields cause saidtarget ion channel to be released from inactivation.
 7. The method ofclaim 1, wherein said plurality of said host cells comprise a voltagesensor selected from the group consisting of a FRET based voltagesensor, an electrochromic transmembrane potential dye, a transmembranepotential redistribution dye, an ion sensitive fluorescent orluminescent molecule and a radioactive ion.